iGEM 2024 — Team cjuh-jlu-china

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Jan.

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Apr.

May

Jun.

Jul.

Aug.

Sept.

Oct.

N0v.

Dec.

Jan.

Feb.

Mar.

Apr.

Wet lab

Model

HP&Edu

Wiki

Presentation

Wet lab

Model

HP&Edu

Wiki

2024.01.02 Sunday

The Recruitment of CJUH-JLU-China

Our PI Xin Hu and Youzhong Wan delivered the basic introduction of
iGEM and the awards of 2023 CJUH-JLU-China team. Team members Saiyu
Luo, Zhongyu Chen, Luohan Ge, He Sun and Ziming Zhong shared their
experience of working in respective team and the standards of taking
part in their groups. This helped the students to understand iGEM
further.

After this promotional lecture, our PI set a series of different
selection requirements to recruit different groups of students who
were interested in experiments , good at biological research and
passionate about synthetic biology.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image1.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image2.png)

2024.01.07 Sunday

The Establishment of CJUH-JLU-China

The members of our team were basically settled, including six
groups:

Experiment,Wiki,Human Practice,Education,Model and Presentation.

The members of Experiment group were encouraged to go through thesis
extensively in the field of reverse onco-cardiology and
onco-cardiology. During the process of brainstorm,each of us come up
with our own ideas and projects.

2023.01.17 Sunday

The First IGEM Team Meeting

This was the first time we held a meeting with all members in our
team, members in Experiment group shared individual project ideas.

Sirui Dong introduced a project about ERK inhibition system based on
the indentification of miR-21. Expecting to inhibit heart disease
related tumors by inhibiting the function of specific miRNA . He
alos introduced LIRA for the project for the first time.

Saiyu Luo was inspired by IISER-TVM, She introduced a medical system
to deliver BC OMV and HF OMV by extracellular vesicle of Escherichia
coli, which can inhibit breast cancer and Cardiac hypertrophy.

Zhongyu Chen designed a miRNA-targeted therapeutic platform and a
negative feedback system for bacterial targeting and tumour cells
killing. By direct killing and indirect miRNA inhibition, this can
prevent and treat tumors caused by cardiovascular diseases.

Qiyu Tan introduced the Janus Plan project which was inspired by
E.coli Nissle 1917 (EcN). His idea focused on the NRG-1/ErbB
pathway. At the same time, he applied exogenous hNRG-1 to promote
cardiomyocyte proliferation that has a tumour-promoting effect, and
the treatment of tumours (e.g.Trastuzumab ) through the block of
ErbB2, leads to drug-induced heart failure.

Yuehtong Ge proposed the rapid detection method based on
miRNA-107.She regarded the miRNA as a biomarker with the aim of
conveniently assessing the cancer risk of patients with heart
disease. This idea came out from last year iGEM project.

Huixin Liang proposed a project about intelligent drug-loading
micro-robot to thereby improve breast cancer screening rates in
hypertension postmenopausal women , which initiates apoptosis and
drug release by sensing the microenvironment through hormone and
enzyme activities.

Rui Yao proposed a new method to produce IL-1β Fab' antibody through
vitro expression, which can reduce the risk of cancer in patients
with heart disease.

Yuxiao Qin proposed a drug based on adenovirus Sh-Sirt3 to reduce
cardiac and tumour pathogenesis and enhance drug resistance
susceptibility. She introduced a suicide switch to enhance the
security of the drug.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image3.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image4.png)

2024.01.26 Friday

1st brainstorm

This meeting of Exp group started with reports about the further
project conceptions.

Qiyu Tan reported the use of E.coli Nissle 1917 in previous IGEM
teams, confirmed the safety and efficacy of EcN, and elucidated the
therapeutic mechanism of NN in heart failure and tumour.

Sirui Dong came up with the primary thought of Heartecho based on
the RNA recognition system of LIRA. He expected to build LIRA parts
with the function of recognising miRNA-21 to regulate the release of
ERK. This ERK can inhibit the relaese of drugs, thus achieving the
treatment of reverse cardio-oncology. However, PI pointed out that
miRNAs also have physiological functions without heart disease, and
the sensitive OR gate design can cause side effects.

Saiyu Luo improved the vector and added the cardiac-specific
promoter cTnT、tumour-specific promoter Cox2 in front of the
expression parts to increase the drug safety.

Zhongyu Chen improved the design of the microenvironmental sensing
module by integrating in vitro transcription into intracellular
parts. Focusing on safety and targeting ability.

Yuehtong Ge added signal cascade link to the project and added
osteoblastin and apelin as biomarkers.

Huixin Liang changed the details of the project and focused on the
specific mechanisms of CD11b+Ly6G-Ly6Chi monocytes and the
CXCL13-CXCR5 axis.

Yuxiao Qin changed the direction of the project and designed to use
the overexpression of HER2/neu as a marker to initiate the
expression of trastuzumab. This can achieve the dual effect of
simultaneously treating breast cancer and reducing the damage to the
heart caused by tumor drugs.

Rui Yao changed the the project. through the functional modification
of exosomes and put into the human body.proposed to treat myocardial
infarction and inhibited the migration of prostate cancer. through
the functional modification of exosomes and put into the human body.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image5.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image6.png)

2024.01.31 Wednesday

2nd brainstorm

In the second Exp group meeting, PI Xin Hu and PI Youzhong Wan
identified two directions for the next experimental design of Exp
:HeartEcho and Janus Plan, and the two projects were carry on in
parallel.

Janus Plan: Qiyu Tan, Saiyu Luo, Rui Yao, Yiheng Shan

HeartEcho: Sirui Dong,Zhongyu Chen, HuixinLiang, Yuetong Ge, Yuxiao
Qin

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image7.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image8.png)

2024.02.02 Sunday

Further Discussion on HeartEcho and Janus Plan (1)

HeartEcho project team:

Sirui Dong introduced the overall project design. After a week of
brainstorm, the HeartEcho project team decided to change the
recognition criteria, and use the dysregulation of miRNA as the
initiating signal to function directly. This help them to design two
AND gate [1,2].

[1] Synthetic RNA-Based Immunomodulatory Gene Circuits for Cancer
Immunotherapy

[2] Synthesizing AND gate minigene circuits based on CRISPReader for
identification of bladder cancer cells

The first design used BS autodegradation sites for logic
circuit-like gene regulation

The second design utilises the CRISPR system: the generation of gRNA
and Cas9 corresponds to the recognition of different miRNAs. When
both miRNAs exists,the CRISPR system is completed and the targeted
therapeutic function can be started.

Janus plan project team:

Qiyu Tan introduced the overall project design within the
experimental group.They showedindividual views on gene vectors,
microbial cells , drug release, etc.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image9.png)

2024.02.05 Wednesday

Further Discussion on HeartEcho and Janus Plan (2)

Janus plan project team:

Qiyu Tan introduced the heart failure biomarker: NT-proBNP.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image10.png)

HeartEcho project team:

The group investigated the potential targets of miR-130b-3p and
miR-21 based on previous designs, and initially clear that miRNA
influence tumour progression through heart disease. The AND gate
design was also found to have the capacity to be further set as a
part that specifically recognises the two target miRNAs.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image11.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image12.png)

2024.02.18 Tuesday

Further Discussion on HeartEcho and Janus Plan (3)

Janus plan project team:

Saiyu Luo introduced us the design of NN release system based on
NT-proBNP and furin protease, which can inhibit the hydrolysis of NN
at high NT-proBNP expression, so as to release NN. Qiyu Tan put
forward two proposals about testing miRNAs in vivo.

Members of the team reported Salmonella which can carry out NN and
the system-PVC which can deliver proteins to the intracellular
precisely.

HeartEcho project team:

Zhongyu Chen designed a diagnostic model of Heartecho based on the
previous AND gate, which is combined with the repressor. After
discussion, the group members changed direction of the project to
the diagnostic, dedicated to develope a diagnostic system for
reverse onco-cardiology.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image12.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image14.png)

2024.02.21 Friday

Further Discussion on HeartEcho and Janus Plan (4)

The members of the experimental group had fourth group meeting
online to debrief and share individual ideas about the project with
each other.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image15.png)

2024.2.25 Tuesday

Seminar of HeartEcho and Janus plan

CJUH-JLU-China members gathered at the China-Japan Union Hospital of
Jilin UniversityC for the fifth group meeting.

HeartEcho project team:

The group members clarified the project as early diagnosis for
tumour risk in patients by identifying overexpression of two miRNAs
and tumour markers. According to this they designed two programs:
One is the AND gateway detection based on BS degradation locus and
outputting the results by using trans-cleavage fluorescence of
CRISPR/Cas13a, the other is the use of enzymatic cascade reaction to
achieve identification of multiple markers and output using the
cell-free system for output.

PI Wan and PI Hu confirmed our progress, but also pointed out that
the focus should be on finding AND gate methods that can recognise
two miRNAs at the same time to further simplify the design; The
selection of miRNA should be further interfaced with the mod group,
and the database should be used to analyse the identification of
miRNA targets.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image16.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image17.png)

Janus plan project team:

We cleared the direction of the project: to regulate the steady
release of anti-heart failure drugs in real time by long-term
monitoring of heart failure markers in cancer chemotherapy patients.
In order to mitigate the cardiotoxic effects due to chemotherapy.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image18.png)

2024.3.3 Sunday

Further Discussion on HeartEcho and Janus Plan (5)

The experiment team members and PI gathered at the China-Japan Union
Hospital of Jilin University for the first meeting of the new
semester.

HeartEcho project team:

The group indentified the project as the detection of two miRNAs and
improved this to get three AND gate strategies for recognising
miRNAs:

①Two OR gates of LIRA were directly expected to achieve the dection
of two miRNAs;

②Non-chemically regulated Repressor was used to connect two AND
gates;

③RNA secondary stem-loop structures(Hairpin) was used to achieve
simultaneous two miRNAs recognition by AND gate strategies.

PI Wan clarified the identification of miRNA by AND gate and
expected to simplify it by using two miRNAs .

For miRNA screening, we found four miRNAs, miR-21, miR-130b,
miR-155, and miR-29a, according to the thesis. [1] Based on the PI's
suggestions and the screening results of the model group, we
continued to expand the scope to look for the intersection of heart
disease and tumour high-expression miRNAs and to confirm their
effective targets.

[1]CVD phenotyping in oncologic disorders: cardio-miRNAs as a
potential target to improve individual outcomes in revers
cardio-oncology,

Janus plan project team:

Qiyu Tan summarised the recent group work and reported the improved
project, which described the mechanism of cardiotoxicity, generation
and release of anthracyclines. The modified project was unanimously
approved.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image19.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image20.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image21.png)

2024.3.8 Friday

Seminar of Janus Plan

Janus Plan project team:

Saiyu Luo used PROTAC technology to redesign the NN release system,
which can regulate NN release more precisely. Yiheng Shan introduced
us about the cTnT (cardiac troponin)-specific promoter, which
controls the site of gene expression. Rui Yao improved the details
of the detection system about heart failure biomarkers.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image22.png)

2024.3.10 Sunday

Further Discussion on HeartEcho and Janus Plan (6)

Heartecho project team:

We further clarified the positioning of the project:

①Problems to be addressed:Idntifying people at high risk of tumours
in patients with heart disease.

②Core:The AND system of identifying two miRNAs.

According to LIRA, the team members proposed several ways to
implement AND gate:

①The recognition fragment was replaced with the stitching of miR1
and miR2, and expression was initiated only if both RNAs were
recognised.

③The shunt of two LIRA AND gates.

After brainstorm and considering the feasibility, stability, and
off-target issues, the first design option was chosen
comprehensively.

Further advancing miRNA screening, members of the experiment group
reported that miR-155, miR-130b-3p, miR-21-5p, and miR-29 all
inhibit tumor cell growth,which did not meet our expectations.

Based on screening results from model groups in TCGA and GEO
databases and on the recommendation of our PI, we decided to
retrieve miR-590-5p, which is highly expressed in heart failure,
pulmonaryhypertension, and multiple tumors. The search strategy is
improved by comparing different diseases.

At the same time, we prepared a proof of concept for LIRA based on
the recommendations of our PI: We worked with the Model group to
perform computer structure simulation using NUPACK software.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image23.png)

Janus Plan Project team:

As the subject of the project is clear : To reduce the cardiotoxic
effects of cancer chemotherapy, the project has been renamed to
“V-oncocardio”,Qiyu Tan designed the vitro detection of two
microRNAs with the AND gate strategy and reported in the group
meeting.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image24.png)

2024.3.17 Sunday

Further Discussion on HeartEcho and V-oncocardioproject (7)

Heartecho project team:

We reported progress on sequence structure: we cleared H01 and H05
sequence in the LIRA library, and successfully recurred structures
about the LIRA on NUPACK and RNA Structure, which requires
relatively low free energy and 55-65°C microenvironment.

PI confirmed our progress and agreed to start the proof-of-concept
experimental design, while continuing to simulate the LIRA structure
of the AND gate, and continue to work with the model group to screen
for miRNA targets.

V-oncocardio project team:

Saiyu Luo briefly introduced the experimental workflows and reported
the results of the recent literature review on BNP promoter, and put
forward the idea of using the BNP promoter. Rui Yao reported the NN
expression system. PI Wan thought that NN expression should be based
on the BNP promoter, which pointed out the direction for our
subsequent research.

The experimental workflow of BNP promoter was briefly introduced by
Saiyu Luo, and a review of recent literature on BNP promoter was
reported. Rui Yao reported on the NN expression system. PI Wan
thought that NN expression should be based on the BNP promoter,
which pointed out the direction for our subsequent research.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image25.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image26.png)

2024.3.23 Saturday

Seminar of V-oncocardioproject

V-oncocardio project team:

We optimised the BNP promoter based on the existing literature, and
reported the design of a shorter BNP promoter and gave the shorter
sequence to the group. And we extensively reviewed the literature
about NN autocrine implementation and completed AAV build process .

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image27.png)

2024.3.24 Sunday

Project Finalisition

At this group meeting, PI Hu and PI Wan hammered out this year's
iGEM project ----HeartEcho. The two groups were merged.

Saiyu Luo, Sirui Dong, Huixin Liang, and Yuyun Qin completed the
proof-of-concept experiment design plan for the H01/H05 sequence.
Yuetong Ge clarified the paper-based cell-free as the output module
of the project system.

Structural validation of two Input AND gates was also completed this
week, and the structures of both NUPACK and RNA Structure validated
our analysis and assumption about the sequences.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image28.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image29.png)

2024.3.31 Sunday

Design experiments about Proof-of-concept and outline the framework
of Parts

Under the guidance of our PI, Saiyu Luo, Sirui Dong, Huixin Liang
and Yuxiao Qin optimised the construction of the target plasmid and
detailed experiment steps.

About Cell-free system,Yuetong Ge made experimental workflows and
design verification experiment about cell-free
transcription-translation system.Rui Yao and Yiheng Shan reviewed
the progress on miRNA.

About Parts, Saiyu Luo and Qiyu Tan summarized the plasmid
construction and Parts Description, and they drafted Parts Overview.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image30.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image31.png)

2024.4.6 Saturday

Design LIRA structure,order plasmids and investigate miRNA

We cooperated with Model Group and reported. Zhongyu Chen designed
the single-arm LIRA under differrnt Stem/Loop Ratio, and compared
their stability according to the free energy.

About the proof-of concept , Saiyu Luo and Yuxiao Qin summarized
them all and orderd the plasmids; Huixin Laing and Sirui Dong
improved the detailed experiments and checked the list of reagents
to be ordered; Yuetong Ge completed the framework of experimental
design related to the cell-free system.

Rui Yao and Yueting Guo conducted extensive literature research
about miR-223-3p and miR-18a-5p, which express highly in myocardial
infarction and atherosclerosis, and can significantly contribute to
gastric cancer.

In terms of paperwork, Qiyu Tan completed the preliminary framework
of the parts page, and Dong Sirui completed the draft of the Chinese
version of the February and March notebook for the Heartecho
project.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image32.png)

2024.4.14 Sunday

Clarify the application and summarize the cell model

Zhongyu Chen,Sirui Dong and Qiyu Tan improved the general
introduction about the project and clarified the background,
solution and application scenarios of the project.

About the experiment design, Saiyu Luo and Yuetong Guo completed the
description and notebook about the color reaction of digestion based
on the Cell-free system.Huixin Liang drafted the protocol about the
proof-of-concept.

About the background investigation, Yueting Guo reviewed the
literature related to the cell model of atherosclerosis; Rui Yao
organized detailed information about the regulation of miR-223-3p in
atherosclerosis genes.

Our PI pointed out the lack of project basis, and suggested that we
should read the literature extensively to clarify the reason why we
chose specific heart disease and cancer as the target of our project
from an epidemiological perspective.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image33.png)

2024.4.21 Sunday

Discussion in-depth about reverse onco-oncology cardiology

We explain the relationship between heart disease and cancer from
the epidemiological perspective. We respectively looked up the
realtionship between atherosclerosis, myocardial infarction, heart
failure , hypertensive and cancer.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image34.png)

2024.4.27 Saturday

Enquiry color reaction and laboratory safety training

Zhongyu Chen summarised the link between atherosclerosis and lung
cancer; Yurtong Ge enquiried the reagents with excellent colour
rendering in the liquid system according to the suggestions of our
PI.

All members of experiment group traind together on laboratory
safety.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image35.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image36.png)

2024.5.12 Sunday

Retrace the project background and summarize the Experimental models

We retraced the background of the project: Sirui Dong, Qiyu Tan and
Saiyu Luo worked together to sort out the development of reverse
oncology cardiology and completed the draft of the description. In
the background research of cardiology, Huixin Liang and RuiYao
reviewed the experimental models of hypertension and atherosclerosis
respectively, while Yutong Ge and Yueting Guo reviewed the
background knowledge of hypertension.

About the screening of miRNA, Yuxiao Qin clarified the role of
microRNA208 in CAD, AMI, ACS and the induction mechanisms of
pancreatic and hepatocellular carcinoma.

About the paperwork, Qiyu Tan completed the manuscript about the
proof-of-concept; Saiyu Luo, Yueting Guo and Yuetong Ge accomplished
the preliminary graft of safety form.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image37.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image38.png)

Experiment

This month, we began to conduct experiments. For ease of recording,
we numbered plasmids 1-35 according to the following table.

![](https://static.igem.wiki/teams/5038/update/img-5-12.webp)

After we received these plasmids and bacteria , we made an overnight
liquid culture and the next day we performed a miniprep or maxiprep
to isolate and quantify the plasmid DNA

For ease of marking,We use “-BL21”as the plasmids' suffix to
indicate corresponding bacteria, we use“→BL21/Trans1-T1”as the
plasmids' suffix to indicate transformation and the bacteria
transformed the corresponding plasmids

For example: ①-BL21 : BL21 containing plasmid① ;②→BL21:Transform
plasmid② to BL21 or BL21 that transformed ② into.

We first proved the concept on plasmids①②⑨⑩ to initially validate
the possibility of LIRA implementation.

2024.05.13 Monday

Outline:

1. Preparation of LB liquid medium and Ampicillin (Amp)/ Kanamycin
(Kan) solid medium [Saiyu Luo][Sirui Dong]

2. Preparation of miniprep plasmids ①②⑨⑩ [Saiyu Luo][Sirui Dong]

①H01 with lac operator-pET-15b ( pET-15b-lacO-H01-linker-EGFP)

②H05 with lac operator-pET-15b (pET-15b-lacO-H05-linker-EGFP)

⑨Input 01 with lac operator-pCOLADuet-1 (pCOLADuet-lacO-input01)

⑩Input 05 with lac operator-pCOLADuet-1 (pCOLADuet-lacO-input05)

Detailed experiments

I. LB liquid medium configuration [Saiyu Luo][Sirui Dong]

LB liquid medium configuration followed the protocol

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image40.png)

1. Bring volume to 2L total by topping up with RO water.

2. Mix until powder is fully dissolved

3. Aliquot the medium into conical flasks, 200mL each.

4. Autoclave on liquid cycle for 20 min ,121℃.

5. Store at room temperature for usage.

II. Solid medium configuration [Saiyu Luo][Sirui Dong]

Solid medium configuration followed the protocol

1. Wash two small conical flasks.

2. Weigh 1.5g agarose, and put it into a small conical flasks
(250mL). Repeat once.

3. Take the above prepared LB medium and dispense about 100mL to
each of the conical flasks containing agarose, seal and shake well;

4. After autoclaving, leave to room temperature.

5. Take 6 petri dishes, labelled "Kan+"; take another 6 petri
dishes, labelled "Amp+".

6. Transfer the conical flask to the ultra-clean table, set it at
room temperature, and add Kan after cooling . the antibiotic
concentration of the original tube is 100mg/mL, and add it to the
medium according to the volumetric ratio of 100μL/100mL, and the
final concentration of antibiotics within the medium is 100μg/mL

7. Pouring plates. A total of 12 plates were made, which were
half-covered with petri dish lids and were set until the medium
solidified. Then sealed with a sealing film.

III. Culture overnight [Saiyu Luo][Sirui Dong]

Remove the bacteria to the 15mL centrifuge tubes containing
LB-Amp/Kan, and left them to shake overnight in the incubator at
37˚C .

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image41.png)

We also spread Glycerol Stock DH5α (Oringinal tube)with the ordered
plasmid①②⑨⑩ on the plate for futher culture.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image42.png)

Handwriting

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image43.png)

2024.05.14 Tuesday

Outline:

1. Miniprep①②⑨⑩ (TlANprep Mini Plasmid Kit) [Qiyu Tan] [Yuetong Ge]

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image44.png)

①H01 with lac operator-pET-15b (pET-15b-lacO-H01-linker-EGFP) (DH5α)

②H05 with lac operator-pET-15b (pET-15b-lacO-H05-linker-EGFP) (DH5α)

⑨Input 01 with lac operator-pCOLADuet-1 (pCOLADuet-lacO-input01)
(DH5α)

⑩Input 05 with lac operator-pCOLADuet-1(pCOLADuet-lacO-input05)
(DH5α)

2. Stored bacteria [Qiyu Tan] [Yuetong Ge]

3. Digestion and Agarose Gel Electrophoresis Verification [Qiyu Tan]
[Yuetong Ge]

We validated the digestion of plasmid①②⑨⑩(Miniperp)

Detailed experiments

I. Stored bacteria [Qiyu Tan] [Yuetong Ge]

II. Miniprep [Qiyu Tan] [Yuetong Ge]

Miniprep Protocol followed: TlANprep Mini Plasmid Kit protocol

Before miniprep ,we firstly used an inoculating wire loop to pick up
some bacteria colonies from a culture plate and inoculate in an 15mL
centrifuge tube containing the LB-Amp/Kan.

1. Label 1.5mL Eppendorf tubes.

2. Fill the tubes with the bacterial culture and centrifuge at 12000
rpm for 1 minute, then discard the supernatant.

3. Repeat step 2 multiple times until all 5 mL of the bacterial
culture is centrifuged.

4. Add 150µL P1 solution, vortex until the solution turns pink.

5. Add 150µL P2 solution, gently invert 6-8 times.

6. Add 350µL P5 solution, immediately invert 12-20 times to mix
until a white flocculent precipitate forms, then centrifuge at 12000
rpm for 1 minute.

7. Prepare an adsorption column by inserting it into a collection
tube.

8. Using a yellow pipette tip nested in a white pipette tip,
transfer the supernatant to the adsorption column with a 200 µL
pipette.

9. Centrifuge at 12000 rpm for 1 minute

10. Discard the supernatant and retain the adsorption column.

11. Add 300 µL PWT solution, centrifuge at 12000 rpm for 1 minute.

12. Discard the waste liquid from the collection tube and replace
the tube.

13. Centrifuge the empty tube at 12000 rpm for 1 minute.

14. Insert the adsorption column into a new Eppendorf tube, add 50
µL ddH2O, centrifuge at 12000 rpm for 1 minute, repeat this step
twice.

15. Measure the concentration.(below)

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image45.png)

III. Digestion verification [Qiyu Tan] [Yuetong Ge]

● We used enzyme EcoRI - HF and XbaI with rCutSmartBuffer.

● We digest them at 37℃ for 1 hour.

● The following volumes were added in the mixture:

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image46.png)

IV. Agarose Gel Electrophoresis

Agarose Gel Electrophoresis followed the protocol

1. Measure 1g agarose.

2. Mix agarose powder with 120mL 1xTAE in a microwavable flask

3. Microwave for 1-3 min until the aga rose is completely dissolved

4. Let agarose solution cool down to about 50 °C.

5. Add Gelstain , usually about 20μL per 150 mL gel

6. Pour slowly to avoid bubbles which will disrupt the gel. Any
bubbles can be pushed away from the well comb or towards the
sides/edges of the gel with a pipette tip.

7. Place newly poured gel at room temperature for 40 mins, until it
has completely solidified.

Note:

● Digested plasmid samples were loaded into the gel as the table
below.

● The concentration of the gel is 1%.

● 2μL of 6X loading buffer were added to all samples.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image47.png)

unit: μL

● Run the gel at 135V for about 40 minutes and then imaged it with
UV light.

Analysis:

There were no visible bands,We inferrd that the loading amount is
too low, so we expected to increase the loading amount and repeat
the experiment again.

Handwriting

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image48.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image49.png)

2024.05.15 Wednesday

Outline:

The Digestion results of miniprep ①②⑨⑩ were not satisfactory, We
inferred that it is because of the low quantity of miniprep. We
planned to perform maxiprep to verify the plasmids.

1. Maxiprep ①②⑨⑩ [Saiyu Luo] [Qiyu Tan][Rui Yao]

①H01 with lac operator-pET-15b (pET-15b-lacO-H01-linker-EGFP) (DH5α)

②H05 with lac operator-pET-15b (pET-15b-lacO-H05-linker-EGFP) (DH5α)

⑨Input 01 with lac operator-pCOLADuet-1 (pCOLADuet-lacO-input01)
(DH5α)

⑩Input 05 with lac operator-pCOLADuet-1(pCOLADuet-lacO-input05)
(DH5α)

Detailed experiments

I. Stored bacteria [Saiyu Luo] [Qiyu Tan][Rui Yao]

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image50.png)

II. Maxiprep ①②⑨⑩ [Saiyu Luo] [Qiyu Tan][Rui Yao]

Protocol followed: EndoFree maxi Plasmid Kit protocol

1. Harvest 200 mL overnight cultured bacterial cells by centrifuging
at 8,000 rpm for 10 min at room temperature (22°C), and then remove
all traces of supernatant.

2. Try to remove all traces of supernatant, use clean paper tissue
to absorb the fluids inside the tube wall.

3. Resuspend pelleted bacterial cells in 8 mL Buffer P1. The
bacteria should be resuspended completely by vortex or pipetting up
and down until no cell clumps remain.

4. Add 8 mL Buffer P2 and mix thoroughly by inverting the tube 7
times, then incubate at room temperature for 5 min.

5. Add 8 mL Buffer P4, and mix immediately and thoroughly by gently
inverting 6-8 times, until the whole solution become cloudy.
Incubate at room temperature for 10 min. Centrifuge for 5-10 min at
8,000 rpm, the white material should be in the bottom of the
centrifuge tube. Transfer the supernatant into a Filtration CS1.
Gently insert the plunger into the Filtration CS1 and filter the
cell lysate into a new 50 mL tube.

6. Column equilibration: place a Spin Column CP6 into a 50 mL
Collection Tube and add 2.5 mL Buffer BL to Spin Column CP6.
Centrifuge for 2 min at 8,000 rpm. Discard the flow- through, and
place Spin Column CP6 into the same Collection Tube

7. Add 0.3×volume isopropanol to the cleared lysate, mix completely
by reverting upside and down and then transfer all solution to the
Spin Column CP6.

8. Centrifuge for 2 min at 8,000 rpm. Discard the flow-through and
place the Spin Column CP6 back into the same Collection Tube.

9. Add 10 mL Buffer PW to the Spin Column CP6 and centrifuge at
8,000 rpm for 2 min. Discard the flow-through and place the Spin
Column CP6 back into the same Collection Tube.

10. Repeat step 9.

11. Add 3 mL 100% ethanol to the Spin Column CP6 (put the CP6 in a
Collection Tube). Centrifuge for 2 min at 8,000 rpm, discard the
flow-through.

12. Put Spin Column CP6 back to Collection Tube, centrifuge at 8,000
rpm for 5 min for removing residual ethanol.

13. To elute DNA, place the Spin Column CP6 in a clean 50 mL
Collection Tube and add 1-2 mL Buffer TB to the center of the
membrane and incubate 5 min at room temperature, centrifuge at 8,000
rpm for 2 min. Transfer the eluate from 50 mL centrifuge tube to a
clean 1.5 mL centrifuge tube and put at -20 °C for storage.

14. Measure the concentration.(below)

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image51.png)

Handwriting

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image52.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image53.png)

2024.05.17 Friday

Outline:

I. Single Digestion and Agarose gel electrophoresis

[Saiyu Luo] [Huixin Liang] [Yuxiao Qin]

We digest Plasmid①②⑨⑩ after maxiprep in order to verify correctness
of the maxiprep.

①H01 with lac operator-pET-15b (pET-15b-lacO-H01-linker-EGFP) (DH5α)

②H05 with lac operator-pET-15b (pET-15b-lacO-H05-linker-EGFP) (DH5α)

⑨Input 01 with lac operator-pCOLADuet-1 (pCOLADuet-lacO-input01)
(DH5α)

⑩Input 05 with lac operator-pCOLADuet-1(pCOLADuet-lacO-input05)
(DH5α)

II. Sequencing maxiprep Plasmid ①②

①H01 with lac operator-pET-15b
(pET-15b-lacO-H01-linker-EGFP)(Trans1-T1)

②H05 with lac operator-pET-15b (pET-15b-lacO-H05-linker-EGFP)
(Trans1-T1)

Detailed experiments

I. Single Digestion [Saiyu Luo] [Huixin Liang] [Yuxiao Qin]

●We used enzyme EcoRI - HF and XbaI with rCutSmartBuffer.

●We digest them at 37℃ for 1 hour.

● The following volumes were added in the mixture:

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image54.png)

II. Agarose gel electrophoresis[Saiyu Luo] [Huixin Liang] [Yuxiao
Qin]

Agarose Gel Electrophoresis followed the protocol

● Digested plasmid samples were loaded into the gel as the table
below.

● The concentration of the gel is 1%.

● 2μL of 6X loading buffer were added to all samples.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image55.png)

● Run the gel at 135V for about 35 minutes and then imaged it with
UV light.

Analysis of results

Successful in maxiprep plasmid①② , but concentrations are lo

Bands of ⑨⑩Input 01/05 samples were unsuccessful

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image56.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image57.png)

III. Sequencing results are correct

①H01-1

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image58.png)

①H01-2

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image59.png)

②H05

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image60.png)

Handwriting

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image61.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image62.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image63.png)

2024.05.18 Saturday

Outline:

Single/Double Digestion and agarose gel electrophoresis: ①②⑨⑩
maxiprep

[Saiyu Luo] [Huixin Liang] [Sirui Dong]

Because of the low loading amount of ①② single enzyme bands and the
failure of the ⑨⑩ experiments, we increased the volume of samples,
performed duplicate experiments with single digestion, and verified
them by doing double digestion.

①H01 with lac operator-pET-15b (pET-15b-lacO-H01-linker-EGFP)

②H05 with lac operator-pET-15b (pET-15b-lacO-H05-linker-EGFP)

⑨Input 01 with lac operator-pCOLADuet-1 (pCOLADuet-lacO-input01)

⑩Input 05 with lac operator-pCOLADuet-1(pCOLADuet-lacO-input05)

Detailed experiments

I. Single Digestion one more time [Saiyu Luo] [Huixin Liang] [Sirui
Dong]

Because of the low concentration, we increased plasmid quality from
270ng to 2μg

We used enzyme EcoRI - HF and XbaI with rCutSmartBuffer.

The concentration of the gel is 1%.

We digest them at 37℃ for 1 hour.

The following volumes were added in the mixture(2μg)

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image64.png)

II. Double Digestion [Saiyu Luo] [Huixin Liang] [Sirui Dong]

We used enzyme EcoRI & Hpa I and NheI & Hpa I with rCutSmartBuffer.

The concentration of the gel is 1%.

We digest them at 37℃ for 1hour.

The following volumes were added in the mixture:

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image65.png)

Ⅲ. Agarose gel electrophoresis [Saiyu Luo] [Huixin Liang] [Sirui
Dong]

Agarose Gel Electrophoresis followed the protocol

(1)Single Digestion

Digested plasmid samples were loaded into the gel as the table
below.

4μL of 6X loading buffer were added to all samples.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image66.png)

● Run the gel at 135V for about 40 minutes and then imaged it with
UV light.

(2)Double Digestion

Digested plasmid samples were loaded into the gel as the table
below.

4μL of 6X loading buffer were added to all samples.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image67.png)

Run the gel at 135V for about 40 minutes and then imaged it with UV
light.

Analysis of results

Single Digestion verified that plasmid extraction of
①②⑨⑩H01&H05&Input01&Input05 is successful. However, the
concentration was low.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image68.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image69.png)

Double Digestion verified that ①②⑨⑩H01&H05&Input01&Input05 were
successful in maxiprep but at low concentration.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image70.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image71.png)

Handwriting

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image72.png)

2024.5.19 Sunday

Literature seminar about miRNA

For the miR-142-3p、miR-146b-5p、miR-330-5p、miR-210a-3p,which
express highly at the intersection of heart failure, myocardial
infarction and cancer, we had a detailed and extensive
investigation.

MiR-210a-3p expresses highly in PH, HF, CAD and widespread promote
the progress on cancer.

MiR-142-3p is co-expressed at high levels in heart failure and
myocardial infarction. It is recognised to promote renal cell
carcinoma and gastric cancer, despite being investigated as a
broadly tumor suppressor.

miR-146b-5p、miR-330-5p are excluded because of the unsatisfactory
effects in the reverse onco-cardiology.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image73.png)

2024.05.20 Monday

Outline:

1. Transformation: Plasmids①②⑨⑩→ Trans1-T1/BL21

[Saiyu Luo] [Qiyu Tan] [Huixin Liang]

After verifying the success of the maxiprep of plasmid ①②⑨⑩, we
decided to examine the transformation , which is in line with the
process of our system expression.

①H01 with lac operator-pET-15b
(pET-15b-lacO-H01-linker-EGFP)(Amp)→Trans1-T1& BL21

②H05 with lac operator-pET-15b (pET-15b-lacO-H05-linker-EGFP)
(Amp)→Trans1-T1&BL21

⑨Input 01 with lac operator-pCOLADuet-1 (pCOLADuet-lacO-input01)
→Trans1-T1& BL21

⑩Input 05 with lac operator-pCOLADuet-1(pCOLADuet-lacO-input05)
→Trans1-T1& BL21

Detailed experiments

I. Pour plates [Saiyu Luo] [Qiyu Tan] [Huixin Liang]

1. Wash the conical bottle, add 1.5g AGAR and 100mL LB medium (add
LB medium on a super clean table, seal after operation)

2. Heat 30s, repeat several times, and shake between each heat,
until the AGAR is completely dissolved, set until it is not hot

3. Add 100μL Amp to 2 bottles, shake well, pour a total of 11
plates.

4. Add 100μL Kan to 2 bottles, shake well, pour a total of 11
plates.

II. Transformation(8 groups)[Zhongyu Chen][Huixin Liang] [Rui
Yao][Yueting Guo]

Transformation followed the protocol Bacterial Transformation

1.Take competent cells out of -80°C and thaw on ice

2.Remove agar plates from storage at 4°C and let warm up to room
temperature and then incubate in 37°C incubator.

3.Mix 2μL of plasmids (500ng) into 50μL of competent cells in a
microcentrifuge tube. Gently mix them

4.Incubate the competent cell/plasmid mixture on ice for 20 mins.

5.Heat shock each transformation tube by placing the bottom 1/2 of
the tube into a 42°C water bath for 90s.

6.Put the tubes back on ice for 2 min.

7.Add 500μl SOC media (without antibiotic) to the bacteria and grow
in 37°C shaking incubator for 1h.

8.Plate transformation (200 μL) onto a LB agar plate containing the
appropriate antibiotic.

9.Incubate plates at 37°C overnigh

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image74.png)

Analysis of results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image75.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image76.png)

III. Pick colonies from the transformed plates and shake

①H01 with lac operator-pET-15b(2个) Trans1-T1;BL21

②H05 with lac operator-pET-15b(2个) Trans1-T1

⑨Input01 with lac operator-pCOLADuet-1(1个) BL21

⑩Input05 with lac operator-pCOLADuet-1(2个) Trans1-T1;BL21

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image85.png)

Dosage of systemic antibiotics:

Amp:100mg/mL 100mL LB+100μL Amp

Kan:100mg/mL 100mL LB+100μL Kan

Handwriting

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image86.png)

2024.05.21 Tuesday

Outline:

1. Transformation: ②⑨⑩→BL21/ Trans1-T1

[Zhongyu Chen][Huixin Liang] [Rui Yao][Yueting Guo]

We repeated transformation beaause of the last failure of
transformation that did not grow properly.

②H05 with lac operator-pET-15b (pET-15b-lacO-H05-linker-EGFP)
→Trans1-T1

⑨Input 01 with lac operator-pCOLADuet-1 (pCOLADuet-lacO-input01)
→Trans1-T1

⑩Input 05 with lac operator-pCOLADuet-1(pCOLADuet-lacO-input05)
→Trans1-T1& BL21

Detailed experiments

I. Stored Bacteria and culture to shake

·Add 800μL bacterial solution to 200μL glycerin and frozen at -80℃

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image87.png)

We removed them in culture tubes containing LB-Amp/LB-Kan,left them
to shake in the incubator at 37˚C overnight.

Add 1mL bacterial solution to 200mL Amp/kan medium, shake at 220rpm
at 37℃ overnight

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image88.png)

II. Transformation ②⑨⑩→BL21/ Trans1-T1

Transformation followed the protocol Bacterial Transformation

Strangely, ②⑨⑩→Trans1-T1/BL21(DE3) grew on the plate, but did not
grow at all in liquid culture.

Although all colonies grew after the last transformation, there were
individual colonies that grew abnormally after the expanded culture,
so the experiment was repeated for verification as follows.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image89.png)

Note: We used LB-Amp/Kan plates.

Analysis of results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image90.png)

Handwriting

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image91.png)

2024.05.22 Wednesday

Outline:

1. Maxiprep for plasmid ①②⑨⑩

[Saiyu Luo][Zhongyu Chen][Qiyu Tan][Huixin Liang][Sirui Dong]

After growth of the transformed colonies, we performed plasmid
maxiprep of bacteria transformed to BL21 and Trans1-T1 to validate
the success of the transformation.

①H01 with lac operator-pET-15b (pET-15b-lacO-H01-linker-EGFP)

②H05 with lac operator-pET-15b (pET-15b-lacO-H05-linker-EGFP)

⑨Input 01 with lac operator-pCOLADuet-1 (pCOLADuet-lacO-input01)

⑩Input 05 with lac operator-pCOLADuet-1(pCOLADuet-lacO-input05)

Detailed experiments

I. Maxiprep ①②⑨⑩

[Saiyu Luo][Zhongyu Chen][Qiyu Tan][Huixin Liang][Sirui Dong]

Protocol followed: TlANprep Mini Plasmid Kit protocol

2024.05.23 Thursday

Outline:

Maxiprep one more time

[Huixin Liang][Rui Yao][Yuetong Ge]

②H05 with lac operator-pET-15b (pET-15b-lacO-H05-linker-EGFP) (Amp)
(DH5α)

②H05 with lac operator-pET-15b (pET-15b-lacO-H05-linker-EGFP) (Amp)
(→BL21(DE3)) (1)

②H05 with lac operator-pET-15b (pET-15b-lacO-H05-linker-EGFP) (Amp)
(→BL21(DE3)) (1)

⑩Input05 with lac operator-pCOLADuet-1 (Kan) (DH5α)

Detailed experiments

I. Maxiprep [Huixin Liang][Rui Yao][Yuetong Ge]

Protocol followed: TlANprep maxi Plasmid Kit protocol

Measure the concentration.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image92.png)

2024.05.24 Friday

Outline:

1. Maxiprep:⑨In01 Lac+ K+ Trans1-T1 maxiprep [Saiyu Luo]

After completing the maxiprep , we performed Digestion validation

2. Single Digestion and Agarose gel electrophoresis: [Saiyu Luo][Rui
Yao] [Yueting Guo]

①H01 with lac operator-pET-15b
(pET-15b-lacO-H01-linker-EGFP)(BL21、Trans1-T1)

②H05 with lac operator-pET-15b (pET-15b-lacO-H05-linker-EGFP)
(BL21、Trans1-T1)

⑨Input 01 with lac operator-pCOLADuet-1
(pCOLADuet-lacO-input01)(BL21、Trans1-T1)

⑩Input 05 with lac operator-pCOLADuet-1(pCOLADuet-lacO-input05)
(BL21、Trans1-T1)

3. Transformation :③④⑨⑩→Trans1-T1 [Saiyu Luo]

At the same time, the plasmids③④⑨⑩we ordered arrived, and we
transformed them followed by the plasmids①②⑨⑩

③H01 without lac operator-pET-15b(pET-15b-H01-linker-EGFP)

④H05 without lac operator-pET-15b(pET-15b-H05-linker-EGFP)

⑤Input 01 without lac operator-pCOLADuet-1(pCOLADuet-input01)

⑥Input 05 without lac operator-pCOLADuet-1(pCOLADuet-input05)

Detailed experiments

I. Maxiprep ⑨ [Saiyu Luo][Rui Yao] [Yueting Guo]

Protocol followed: TlANprep maxi Plasmid Kit protocol

Measure the concentration.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image93.png)

II. Single Digestion [Saiyu Luo]

●We used enzyme EcoRI - HF and XbaI with rCutSmartBuffer.

●We digest them at 37℃ for 30minutes.

● The following volumes were added in the mixture(2μg):

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image94.png)

Agarose Gel Electrophoresis followed the protocol

Digested plasmid samples were loaded into the gel as the table
below.

The concentration of the gel is 1%.

4μL of 6X loading buffer were added to all samples.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image95.png)

unit: μL

Analysis of results

Single Digestion verified that H01&H05&Input05 plasmid extraction
successfully, except Input05 which had lower concentration and
Input05 had heterozygous bands

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image96.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image97.png)

III. Transformation :③④⑨⑩→Trans1-T1 [Saiyu Luo]

Transformation followed the protocol Bacterial Transformation

Analysis of results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image98.png)

Handwriting

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image99.png)

2024.05.25 Saturday

Outline:

1. Single/double Digestion and Agarose Gel Electrophoresis
verification :

⑨Input01→Trans1-T1

[Saiyu Luo][Sirui Dong][Huixin Liang][Yuxiao Qin]

⑨Input 01 with lac operator-pCOLADuet-1
(pCOLADuet-lacO-input01)(Trans1-T1)

2. Preparation of maxiprep

[Saiyu Luo][Sirui Dong][Huixin Liang][Yuxiao Qin]

③H01 without lac operator-pET-15b(pET-15b-H01-linker-EGFP)

④H05 without lac operator-pET-15b(pET-15b-H05-linker-EGFP)

⑤Input 01 without lac operator-pCOLADuet-1(pCOLADuet-input01)

⑥Input 05 without lac operator-pCOLADuet-1(pCOLADuet-input05)

Detailed experiments

I. Single Digestion :Input01 Trans1-T1 [Saiyu Luo][Sirui
Dong][Huixin Liang][Yuxiao Qin]

●We used enzyme EcoRI - HF and XbaI with rCutSmart Buffer.

●We digest them at 37℃ for 30min.

● The following volumes were added in the mixture:

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image100.png)

II. Double Digestion

[Saiyu Luo][Sirui Dong][Huixin Liang][Yuxiao Qin]

●We used enzyme EcoRI & Hpa I and NheI & Hpa Iwith rCutSmartBuffer.

●We digest them at 37℃ for 30minutes.

● The following volumes were added in the mixture:

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image101.png)
Ⅲ. Agarose gel electrophoresis [Saiyu Luo] [Huixin Liang] [Sirui
Dong]

Agarose Gel Electrophoresis followed the protocol

(1)Single Digestion

Digested plasmid samples were loaded into the gel as the table
below.

The concentration of the gel is 1%.

4μL of 6X loading buffer were added to all samples.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image102.png)

unit: μL

Run the gel at 135V for about 40 minutes and then imaged it with UV
light.

(2)Double Digestion

Digested plasmid samples were loaded into the gel as the table
below.

The concentration of the gel is 1%.

4μL of 6X loading buffer were added to all samples.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image103.png)

unit: μL

Run the gel at 135V for about 40 minutes and then imaged it with UV
light.

Analysis of results

Single Digestion verified that plasmid extraction of ①②⑨⑩ H01&H05 &
Input01(BL21) &Input05(BL21) is successful. However, the
concentration was low

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image104.png)

Double Digestion verified the success of ①H01&②H05 (Trans1-T1) in
maxiprep.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image105.png)

III. Pick colonies from the transformed plates and shake

[Saiyu Luo][Sirui Dong][Huixin Liang][Yuxiao Qin]

We did this to prepare for the maxiprep tomorrow

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image106.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image107.png)

Handwriting

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image108.png)

2024.05.26 Sunday

Project learning, confirm miRNA and improve cell-free system

Zhongyu Chen reported on the LIRA OR gate of UGM-Indonesia team for
detecting biomarkers in colon cancer, identifying our key issues to
be addressed.

Yuetong Ge improved the functional verification of lacZα about the
cell-free system.

Saiyu Luo searched for potential miR-210-3p, miR-142-3p targets.
54,128 target genes were predicted using mirDIP, miRWalk, miRDB and
analyzed using the TCGA database

Yuxiao Qin and Yuetong Ge clarified that miR-455-3p/5p is highly
expressed in myocardial fibrosis, cardiac hypertrophy and promotes
colon cancer, breast cancer, and melanoma.

QiYu Tan, Huixin Liang, Rui Yao, SiRui Dong, and YueTing Guo
reviewed the literature related to miR-450b-5p, miR-199a-5p,
miR-199b-5p, and miR-576-5p in cardiovascular diseases and cancers
respectively, and excluded them after discussion.

After a month collaborative effort between the experiment group and
Model group, we finally confirmed miR-210-3p and miR-142-3p as our
biomarkers for diagnosis under the guidance of our PI.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image109.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image110.png)

2024.05.27 Monday

Outline:

1. Maxiprep & Concentration measurement: ③④

[Saiyu Luo][Huixin Liang][Rui Yao][Yueting Guo]

③H01 without lac
operator-pET-15b(pET-15b-H01-linker-EGFP)(Trans1-T1)(1)

③H01 without lac
operator-pET-15b(pET-15b-H01-linker-EGFP)(Trans1-T1)(2)

③H01 without lac
operator-pET-15b(pET-15b-H01-linker-EGFP)(Trans1-T1)(3)

④H05 without lac
operator-pET-15b(pET-15b-H05-linker-EGFP)(Trans1-T1)(1)

④H05 without lac
operator-pET-15b(pET-15b-H05-linker-EGFP)(Trans1-T1)(2)

④H05 without lac
operator-pET-15b(pET-15b-H05-linker-EGFP)(Trans1-T1)(3)

Detailed experiments

I. Stored bacteria [Saiyu Luo][Huixin Liang][Rui Yao][Yueting Guo]

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image111.png)

II. Centrifuge bacterial solution [Saiyu Luo][Huixin Liang][Rui
Yao][Yueting Guo]

Pour the bacterial solution into a 50mL centrifuge tube,Using
centrifuge, 5000rpm, 10min, multiple times. The plasmids were later
stored in the -80℃ fridge

III. Maxiprep [Saiyu Luo][Huixin Liang][Rui Yao][Yueting Guo]

Protocol followed: TlANprep maxi Plasmid Kit protocol

·Concentration measurement

Measure twice and take the average

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image112.png)

2024.05.28 Tuesday

Outline:

1. Single/Double Digestion and Agarose Gel Electrophoresis

[Qiyu Tan] [Yueting Guo][Huixin Liang][Rui Yao]

⑨Input 01 with lac operator-pCOLADuet-1(pCOLADuet-lacO-input01)
(Kan)(DH5α) -5.15(1)(2)

⑩Input 05 with lac operator-pCOLADuet-1(pCOLADuet-lacO-input05)(Kan)
(DH5α) -5.15

⑨Input 01 with lac operator-pCOLADuet-1
(pCOLADuet-lacO-input01)(Kan) (BL21) -5.22

⑩Input 05 with lac
operator-pCOLADuet-1(pCOLADuet-lacO-input05)(Kan)(Trans1-T1) -5.22

⑩Input 05 with lac operator-pCOLADuet-1(pCOLADuet-lacO-input05)
(Kan) (BL21) -5.22

⑨Input 01 with lac operator-pCOLADuet-1
(pCOLADuet-lacO-input01)(Kan)(Trans1-T1)-5.24

⑩Input 05 with lac operator-pCOLADuet-1(pCOLADuet-lacO-input05)
(Kan) (DH5α) -5.24

Detailed experiments

I. Single Digestion

[Qiyu Tan] [Yueting Guo][Huixin Liang][Rui Yao]

●We used enzyme XbaI with rCutSmartBuffer.

●We digest them at 37℃ for 30 minutes.

● The following volumes were added in the mixture:

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image113.png)

II. Double Digestion

[Qiyu Tan] [Yueting Guo][Huixin Liang][Rui Yao]

We used enzyme NheI & Hpa I with rCutSmartBuffer.

We digest them at 37℃ for 30 minutes.

The following volumes were added in the mixture:

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image114.png)

III. Agarose Gel Electrophoresis

[Qiyu Tan] [Yueting Guo][Huixin Liang][Rui Yao]

Follow Protocol :Agarose Gel Electrophoresis

● Digested plasmid samples were loaded into the gel as the table
below.

● The concentration of the gel is 1%.

● 2μL of loading buffer were added to all samples.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image115.png)

unit: μL

● Run the gel at 135V for about 45 minutes and then imaged it with
UV light.

(2)Double Digestion

● Digested plasmid samples were loaded into the gel as the table
below.

● The concentration of the gel is 1%. /div>

● 2μL of loading buffer were added to all samples.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image116.png)

unit: μL

● Run the gel at 135V for about 45 minutes and then imaged it with
UV light.

Analysis of results

Bands are basically correct,but some bands of ⑩In05 Lac+ K+
Trans1-T1 are too bright bands, there may be heterozygous bands,
re-examination of the extraction and Digestion.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image117.png)

The bands of the double digestion is basically correct

Seen from the shallow bands, the quality of the plasmid extraction
concentration is poor, and further expirements are needed.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image118.png)

Handwriting

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image119.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image120.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image121.png)

2024.05.29 Wednesday

Outline:

1. Single colonies picking [Saiyu Luo][Zhongyu Chen][Qiyu Tan]

①H01 with lac operator-pET-15b (pET-15b-lacO-H01-linker-EGFP)

②H05 with lac operator-pET-15b (pET-15b-lacO-H05-linker-EGFP)

Detailed experiments

I. Single colonies picking [Saiyu Luo][Zhongyu Chen][Qiyu Tan]

We pick single colonies from the plates that spreaded the bacteria
solution from oringinal tubes on

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image122.png)

2024.05.30 Thursday

Outline:

1. Maxiprep ⑨⑩

[Yueting Guo][Huixin Liang][Yuetong Ge]

Based on the low signal of the sequencing, the maxiprep Plasmid
was performed again

⑨Input 01 with lac operator-pCOLADuet-1
(pCOLADuet-lacO-input01)DH5α(1)

⑨Input 01 with lac operator-pCOLADuet-1
(pCOLADuet-lacO-input01)DH5α(2)

⑩Input 05 with lac
operator-pCOLADuet-1(pCOLADuet-lacO-input05)DH5α(1)

⑩Input 05 with lac operator-pCOLADuet-1(pCOLADuet-lacO-input05)
DH5α(2)

Detailed experiments

I. Maxiprep [Yueting Guo][Huixin Liang][Yuetong Ge]

Protocol followed: TlANprep maxi Plasmid Kit protocol

·Concentration measurement

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image123.png)

Handwriting

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image124.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image125png)

2024.05.31 Friday

Outline:

1. Single/Double Digestion and Agarose gel electrophoresis [Qiyu
Tan][Yuetong Ge]

⑨Input 01 with lac operator-pCOLADuet-1 (pCOLADuet-lacO-input01)
5.31

⑩Input 05 with lac operator-pCOLADuet-1(pCOLADuet-lacO-input05)
5.31

③H01 without lac operator-pET-15b(pET-15b-H01-linker-EGFP) 5.27

④H05 without lac operator-pET-15b(pET-15b-H05-linker-EGFP) 5.27

Detailed experiments

1.Single/Double Digestion [Qiyu Tan][Yuetong Ge]

●We used enzyme EcoRI - HF and XbaI with rCutSmartBuffer.

●We digest them at 37℃ for 1 hour.

● The following volumes were added in the mixture:

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image125.png)

II.Double Digestion [Qiyu Tan][Yuetong Ge]

●We used enzyme EcoRI & Hpa I and NheI & Hpa I with
rCutSmartBuffer.

●We digest them at 37℃ for 30 minutes.

● The following volumes were added in the mixture:

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image126.png)

Ⅲ. Agarose gel electrophoresis [Qiyu Tan][Yuetong Ge]

Agarose Gel Electrophoresis followed the protocol

(1)Single Digestion

● Digested plasmid samples were loaded into the gel as the table
below.

● The concentration of the gel is 1%.

● 4μL of loading buffer were added to all samples.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image127.png)

unit: μL

● Run the gel at 135V for about 40 minutes and then imaged it with
UV light.

(2)Double Digestion

● Digested plasmid samples were loaded into the gel as the table
below.

● The concentration of the gel is 1%.

● 4μL of loading buffer were added to all samples.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image128.png)

● Run the gel at 135V for about 40 minutes and then imaged it with
UV light.

Analysis:

③④H01/H05 bands are basically correct in size.

⑨⑩In01/05 bands are not clear, the quality of plasmid is
problematic, need to explore.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image129.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image130.png)

③④In01/In05;⑨⑩ H01/H05 bands are almost correct in size.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image131.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image132.png)

Handwriting

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image133.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image134.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image135.png)

2024.06.03 Monday

Outline:

1. Transformation : Plasmids⑤⑥→BL21 [Huixin Liang] [Rui Yao][Yuetong
Ge]

⑤Input 01 without lac operator-pCOLADuet-1(pCOLADuet-input01)

⑥Input 05 without lac operator-pCOLADuet-1(pCOLADuet-input05)

II.Sequencing of correctly digested plasmids ③④⑨⑩

③H01 without lac operator-pET-15b(pET-15b-H01-linker-EGFP)

④H05 without lac operator-pET-15b(pET-15b-H05-linker-EGFP)

⑨Input 01 with lac operator-pCOLADuet-1 (pCOLADuet-lacO-input01)

⑩Input 05 with lac operator-pCOLADuet-1(pCOLADuet-lacO-input05)

Detailed experiments

I. Transformation.

[Huixin Liang] [Rui Yao][Yuetong Ge]

Transformation followed the protocol Bacterial Transformation

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image136.png)

Note:

Mark:⑤In01 Lac- K+ DH5α - 475ng/μL;⑥In05 Lac- K+ DH5α -650ng/μL

Plates :(Antibiotic:Amp/Kan)Mark:Input01/05with lac operator(6.3)
(5.29)

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image137-p.jpg)

II. Sequencing of correctly digested plasmids ③④⑨⑩

[Huixin Liang] [Rui Yao][Yuetong Ge]

③H01 without lac operator-pCOLADuet-1(Trans1-T1) 5.27

④H01 without lac operator-pCOLADuet-1(Trans1-T1) 5.27

⑨Input01 with lac operator pCOLA

⑩Input05 with lac operator pCOLA

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image143.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image144.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image145.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image146.png)

Handwriting

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image147.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image148.png)

2024.06.04 Tuesday

Outline:

1. ⑤⑥ of the Transformation coated plates were picked and expanded
for culture.

[Yueting Guo][Qiyu Tan][Sirui Dong][Yuxiao Qin]

2. Sequencing the correct⑤⑥(5.15) before further transformation
(BL21 competent cells). [Yueting Guo][Qiyu Tan][Sirui Dong][Yuxiao
Qin]

⑤Input 01 without lac operator-pCOLADuet-1(pCOLADuet-input01)

⑥Input 05 without lac operator-pCOLADuet-1(pCOLADuet-input05)

Detailed experiments

I. Transformation. Large Concentration Conversion

14:00-17:00;[Yueting Guo][Qiyu Tan][Sirui Dong][Yuxiao Qin]

Due to the unsatisfactory results of the previous transformation, we
increased the concentration of transformed plasmid.

Transformation followed the protocol Bacterial Transformation

Note: The concentration of the plasmids was 2μL(1000ng)

Mark: Input01/05 pCOLA Kan+ Concentration:2μL ≈ 1000ng

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image149.png)

Plasmid

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image150.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image151.png)

Process

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image152.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image153.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image154.png)

Mark

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image155.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image156.png)

2024.06.05 Wednesday

Outline:

1.Centrifugation of ⑤⑥ bacterial fluids(5.29) [Huixin Liang]

2.Sequencing correct ⑤⑥ transformed coated plate picks(5.15), 15 mL
tubes to be determined are both coated plate or evening remote
bacteria. [Huixin Liang]

⑤Input 01 without lac operator-pCOLADuet-1(pCOLADuet-input01)

⑥Input 05 without lac operator-pCOLADuet-1(pCOLADuet-input05)

Detailed experiments

I. Centrifuge bacterial solution

Note: Input01/05 with lac operator(5.29transformation)no signal

Pour the bacterial solution into a 50mL centrifuge tube,Using
centrifuge, 5000rpm, 10min.

The plasmids were later stored in the -80℃ fridge

Centrifuge bacterial solution

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image157.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image158.png)

Storage location

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image159.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image160.png)

1. Sequencing correct ⑤⑥ transformed coated plate picks(5.15)

The sequencing results are unsatisfactory.

2024.06.06 Thursday

Outline:

Maxiprep:Vigorous plasmid maxiprep kit

In order to further improve the efficiency of the maxiprep,we used
Vigorous plasmid maxiprep kit.

[Yueting Guo][Qiyu Tan][Sirui Dong][Yuxiao Qin]

⑤Input 01 without lac operator-pCOLADuet-1(pCOLADuet-input01) 5.15

⑥Input 05 without lac operator-pCOLADuet-1(pCOLADuet-input05) 5.15

⑤Input 01 without lac operator-pCOLADuet-1(pCOLADuet-input01) 5.15

⑤Input 01 without lac operator-pCOLADuet-1(pCOLADuet-input01) 5.15

Detailed experiments

I. Centrifuge bacterial solution

Pour the bacterial solution into a 50mL centrifuge tube,Using
centrifuge, 5000rpm, 10min, multiple times. The plasmids were later
stored in the -80℃ fridge

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image161.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image162.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image163.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image164.png)

II. Maxiprep⑤⑥

Protocol followed: Vigorous plasmid maxprep kit protocol

(1) Plasmid extraction

1. Add 5 mL Buffer l to the aforesaid bacterial centrifuge tubes,
and mix and shake the bacterial precipitate well to disperse it
completely until no flocs exist. Transfer the bacterial suspension
into a 50 mL centrifuge tube.

2. Add 5 mL Buffer ll, gently invert the centrifuge tube 6-8 times,
and leave it at room temperature for 5 min, so that the bacteria are
completely lysed and the solution is clear.

3. Add 5 mL Buffer lll, immediately invert the centrifuge tube 6-8
times and mix well until white flocculent is produced. Leave on ice
for 12 min.

4. Centrifuge the above lysate at 12,000xg for 15 min at 4°C.
Carefully aspirate the supernatant and transfer to a new 50 mL
centrifuge tube.(After centrifugation, the precipitation is not
completely separated from the supernatant, so repeat centrifugation
once)

5. Add 10 mL of isopropanol. Mix well by inversion and leave on ice
for 12 min.

6. Centrifuge at 12,000xg for 10 min at 4°C. Carefully discard the
supernatant, invert and gently drain off the residouble liquid, add
0.5 mL Buffier l to completely dissolve the precipitate clumps
(dissolution can be assisted by gently blowing with a wide-mouth
pipette). Transfer to a new 1.5mL centrifuge tube and leave at room
temperature for 12min.

7. Centrifuge the crude extract of plasmid at high speed for 2min at
room temperature in a desktop centrifuge, and transfer the
supernatant into a new 1.5mL centrifuge tube.

(2) Plasmid purification

1. 0.5 mL of plasmid crude extract add 100 μl Buffer IV (impurity
removal solution A), gently mixing 12,000xg centrifugation for 2min,
the supernatant will be transferred to a new centrifuge tube.

2. Add 100μl of Buffer IV (Impurity Removal Solution A), mix gently,
centrifuge at 12,000xg for 5min and transfer the supernatant to a
new tube.

3. Add 70mL BufferV (Impurity Removal Solution B), mix gently,
centrifuge at 12,000xg for 5 min, transfer the supernatant to a new
centrifuge tube.

4. 0.5mL isopropanol was added, mixed well, and left at room
temperature for 10 min. 12,000xg centrifugation at room temperature
for 10 min, the supernatant was discarded, washed gently with 1mL of
70% ethanol, the liquid was discarded, and the tube was inverted and
cooled for 5 min at room temperature.

(Preparation of 70% ethanol in this step: 14mL 75% ethanol +1mL
water)

5. 0.5mL ddH2O dissolved precipitate (wide mouth pipette gently
blowing to assist dissolution)

6. Add 200mL Buffer VI (Impurity Removal Solution C), mix well and
leave on ice for 20min, centrifuge at 12,000xg for 10min at room
temperature, discard the supernatant, gently add 1m1 of 70% ethanol
to wash twice, and then invert and cool at room temperature for 5-10
min to make the ethanol evaporate completely.

7. Add appropriate amount of dd H2O (250μL) to dissolve the
precipitate (can be shaken in a 37°C water bath to aid dissolution)

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image165.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image166.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image167.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image168.png)

2024.06.07 Friday

Outline:

Preparation of LB liquid medium and Amp + / Kan + solid medium
[Saiyu Luo]

Detailed experiments

I. LB liquid medium [Saiyu Luo]

LB liquid medium configuration followed the protocol

Total volume =2L

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image169.png)

II. Solid medium [Saiyu Luo]

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image170.png)

We autoclaved and placed them in 1008 cabinet

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image171.png)

Preparation of 8 Kan-LB plates (1:2000)

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image172.png)

Store at 4℃ freezer

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image173.png)

2024.07.07 Sunday

The first summer seminar after the final exam

2024.07.08 Monday

Outline:

I.Transformation:⑤⑥⑦⑧ →BL21[Saiyu Luo][Huixin Liang][Yueting
Guo][Rui Yao]

⑤Input 01 without lac operator-pCOLADuet-1(pCOLADuet-input01)

⑥Input 05 without lac operator-pCOLADuet-1(pCOLADuet-input05)

⑦Input 01 without lac operator-pCDFDuet-1(pCDFDuet-input01)

⑧Input 05 without lac operator-pCDFDuet-1(pCDFDuet-input05)

II.Prepare LB liquid medium[Saiyu Luo][Huixin Liang][Yueting
Guo][Rui Yao]

Detailed Experiments

I Transformation:plasmid ⑤⑥⑦⑧ to BL21

[Saiyu Luo][Huixin Liang][Yueting Guo][Rui Yao]

Follow protocol : Transformation

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image174.png)

The concentration of the plasmid >=100ng/μL

Heat-shock at 42℃ for 30s

Add 250μL SOC medium to every tube before shaking for 1h

Place the coated plate under 37℃ for 30min. After the bacterial
solution dried, invert and cultivate overnight.

Discription Picture

Melt the plasmid and competent cells on ice

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image175.png)

Mark the tubes

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image176.png)

Mark the plates of ⑤→BL21

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image177.png)

Mark the plates of ⑥→BL21

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image178.png)

Mark the plates of ⑦→BL21

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image179.png)

Mark the plates of ⑧→BL21

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image180.png)

Results(7.09 observed):

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image181.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image182.png)

⑤→BL21 ⑥→BL21

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image183.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image184.png)

⑦→BL21 ⑧→BL21

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image185.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image186.png)

II.2L LB medium was prepared and ten Kan and str plates were poured

[Saiyu Luo][Huixin Liang][Yueting Guo][Rui Yao]

Followed protocol :Preparation of LB medium and plates pouring

2024.07.09 Tuesday

Outline:

I. Pick single colonies from transformed plates and shake:⑤⑥⑦⑧→BL21

⑤Input 01 without lac operator-pCOLADuet-1(pCOLADuet-input01)

⑥Input 05 without lac operator-pCOLADuet-1(pCOLADuet-input05)

⑦Input 01 without lac operator-pCDFDuet-1(pCDFDuet-input01)

⑧Input 05 without lac operator-pCDFDuet-1(pCDFDuet-input05)

II.Preparation of SOC medium

Detailed Experiments:

I. Pick single colonies from transformed plates and shake:⑤⑥⑦⑧→BL21

[Saiyu Luo][Zhongyu Chen] [Qiyu Tan][Sirui Dong] [Huixin Liang]
[Yueting Guo]

[Yuxiao Qin] [Rui Yao]

Pick single colonies:⑤⑥⑦⑧→BL21 (three of each )and shake (15mL tube,
220rpm, 37℃ overnight shake)

Discription Pictures

pick single colony:⑤→BL21

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image183.png)

pick single colony:⑥→BL21

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image184.png)

pick single colony:⑦→BL21

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image185.png)

pick single colony:⑧→BL21

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image186.png)

Add 5ml LB and put single colonies picked into 15ml tube,and the 3
tube marked by red square were added the wrong antibiotic so they
were discarded.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image187.png)

The 3 tubes marked by red square are the group that we made again

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image188.png)

Shake for 1h

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image189.png)

II.Preparation of SOC medium

[Saiyu Luo][Zhongyu Chen] [Qiyu Tan][Sirui Dong] [Yuetong Ge]
[Huixin Liang] [Yueting Guo]

[Yuxiao Qin] [Rui Yao]

Follow protocol: Preparation of SOC medium

We prepared 100ml 10xSOC medium this time

Discription Picture

We divided the SOC into two 50ml tubes and store them in the -20℃
refrigerator.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image190.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image191.png)

2024.07.10 Wednesday

Outline:

I. Stored bacteria:⑤→BL21[Sirui Dong]

II. Miniprep:⑤⑥⑦⑧→BL21[Saiyu Luo]

III. PCR:⑤⑥ [Saiyu Luo]

IV. Agarose gel electrophoresis:⑤⑥[Saiyu Luo]

⑤Input 01 without lac operator-pCOLADuet-1(pCOLADuet-input01)

⑥Input 05 without lac operator-pCOLADuet-1(pCOLADuet-input05)

⑦Input 01 without lac operator-pCDFDuet-1(pCDFDuet-input01)

⑧Input 05 without lac operator-pCDFDuet-1(pCDFDuet-input05)

Detailed experiments:

I. Stored bacteria[Sirui Dong]

store the bacteria solution ⑤→BL21 shaken overnight lasi night

Add 800μL bacterial solution to 200μL glycerin and froze at -80℃

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image192.png)

II. Miniprep:⑤⑥⑦⑧→BL21[Saiyu Luo]

Follow protocol :TIANprep Mini Plasmid Kit protocol

After the growth of the transformed colonies, we performed plasmid
Miniprep ⑤⑥⑦⑧→BL21 to verify the plasmid has already transformed
into BL21 successfully.

The concentration of plasmids are as follows(unit:ng/μL):

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image193.png)

III. PCR:⑤⑥[Saiyu Luo]

Followed protocol :PCR

we verified the plasmids we performed miniprep by PCR

System preparation

System composition (25μL system) :

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image194.png)

The experimental steps are as follows:

1. Place the extracted plasmid on ice, defrosting ddH2O.

2.Calculate dilution method (dilute to 10ng/μL first, then dilute 40
times)

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image195.png)

unit:μL

3. Mark 6 new EP tubes

4.Add dd H2O to each EP tube, and then add the 3μL stock solution
corresponding to its label for centrifugation

5. Add 78μL H2O into a new EP tube

6.Take 2μL from the previous round of diluted tube and add it to the
corresponding new EP tube (mixed with 78μL water)

7.Centrifuge, so that the water drops on the wall of the tube, and
then mix with the vortex shaker, and then centrifuge

8.Return the original tube and diluent with a concentration of
10ng/μL to the penultimate layer of the -20℃ refrigerator

9.Centrifuge the primer for 5min

10. Take 2EP tubes, mark them as InputR and InputF, and heat DEPC
water (the water is the leftover product of last year).

11. Add 41.7μLDEPC water to InputF, 40.8μL DEPC water to InputR,
centrifuge and mix well

12. Mix 2μL primer with 98μL dd water, mix with vortex shaker, and
put the remaining primer back to the bottom layer of -20℃
refrigerator

13.Take an EP tube marked with mix and add the following liquid
(7xPCR reaction system other substances except the template) : 35μL
5xPrime Buffer 14μL dNTP mix 75.25μL ddH2O 1.75μL prime STAR enzyme
21μL InputF 21μL InputR

The enzyme needs to be thawed on ice, in order to maintain the
stability of the enzyme character and reduce the loss, generally add
the enzyme at the end, and the enzyme is generally stored with
glycerin

14.Centrifuge and mix

15. Divide mix mixture into 6 EP tubes, each tube 24μL, EP tubes
marked 01/05 1,2,3

16. Add 1μL plasmid with a concentration of approximately 250pg/μL
to each EP tube

Program setting

98℃ 1min

98℃ 10s

55℃ 5s Repeat 30 cycles

72℃ 1.5min(Set according to PCR fragments about 1500nt)

72℃ 2min

40℃ hold

Total about 1.5 hours

17.After completion, put the PCR reagent back into the second layer
of the -20℃ refrigerator

18.Turn off the PCR machine after the temperature drops

19.Return the plasmid diluted to 250pg/μL and the primer with
concentration of 2umol/L to the penultimate layer of the -20℃
refrigerator

IV.Agarose gel electrophoresis:⑤⑥[Saiyu Luo]

Follow protocol : Agarose Gel Electrophoresis

The concentration of the gel is 1%

5μL of loading buffer were added to all samples

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image196.png)

Run the gel at 135V for about 30min and then imaged it with UV light

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image197.png)

No distinct stand except marker

2024.07.11 Thursday

Outline:

I.Single/double enzyme digestion:⑤⑥⑦⑧[Saiyu Luo][Qiyu Tan]

II.Agarose gel electrophoresis[Saiyu Luo]

Detailed experiments:

I.Single/double enzyme digestion:⑤⑥⑦⑧[Saiyu Luo][Qiyu Tan]

Because there was no distinct stant except marker shown on the gel
ran yesterday,we hypothesized that this might be the reason of PCR
problem,so we single[Qiyu Tan]and double [Saiyu Luo]digest all the
plasmids preped in 7.10 (⑤⑥⑦⑧) and run agarose gel to verify.

The single enzyme digestion system is as follows

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image198.png)

The double enzyme digestion system is as follows

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image199.png)

Figure

Discription Picture

The material we used

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image200.png)

II.Agarose gel electrophoresis[Saiyu Luo]

Followed protocol :Agarose Gel Electrophoresis

The concentration of the agarose gel is 1%

2μL 6xloading buffer was added in each sample

Run the gel at 135V for 40min

The loading order and loading quantity are shown in the following
table

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image201.png)

unit:μL

Results

Single enzyme digestion

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image202.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image203.png)

Double enzyme digestion

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image204.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image205.png)

2024.07.12 Friday

Outline:

I. Transformation:⑤⑥⑦⑧→BL21 [Saiyu Luo]

⑤Input 01 without lac operator-pCOLADuet-1(pCOLADuet-input01)

⑥Input 05 without lac operator-pCOLADuet-1(pCOLADuet-input05)

⑦Input 01 without lac operator-pCDFDuet-1(pCDFDuet-input01)

⑧Input 05 without lac operator-pCDFDuet-1(pCDFDuet-input05)

Detailed experiments:

I.Transformation:⑤⑥⑦⑧→BL21 [Saiyu Luo]

Due to the wrong result of agarose gel electrophoresis ,we suspect
that the transformation is a failure,so we re-transform ⑤⑥⑦⑧
plasmids to BL21(DE3) competent cells from a new company(Sangon
Biotech) [Saiyu Luo]

Follow protocol:Bacterial Transformation

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image206.png)

Heat-shock at 42℃ for 45s

Add 350μL LB medium per tube before shake for 1h( 220rpm, 37℃)

Place the coated plate at 37℃ for 30min. After the bacterial
solution dried, invert and cultivate overnight.

Discription Picture

The competent cells we used

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image207.png)
Melt the competent cells and plasmids on ice

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image208.png)

Spread 200μL bacteria solution from the transformation on the plates

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image209.png)

Result (7.13 observed)

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image210.png)

Only ⑥→BL21 grows obvious colonies

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image211.png)

2024.07.13 Saturday

Outline:

I.Pick single colonies from the transformed plates and
shake:⑥→BL21[Saiyu Luo]

II.Bacteriological conservation: ⑥→BL21[Saiyu Luo]

⑥Input 05 without lac operator-pCOLADuet-1(pCOLADuet-input05)

Detailed experiments:

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image212.png)

I.Pick single colonies from the transformed plates and
shake:⑥→BL21[Saiyu Luo]

Dosage of systemic antibiotics:

Amp:100mg/ml 100ml+100μLAmp

Discription Picture

Put single colonies we picked into 15ml tubes

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image213.png)

II.Stored the bacteria ⑥→BL21[Saiyu Luo]

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image214.png)

Add 800μL bacterial solution to 200μL glycerin and frozen at -80℃

Discription Picture

Store the bacteria

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image215.png)

The remaining bacterial solution was stored in a 4℃ refrigerator

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image216.png)

The stored bacteria solution was stored in the -80℃ refrigerator

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image217.png)

2024.07.14 Sunday

The experimental group had a second seminar.

2024.07.15 Monday

Outline:

I. Shake and store the bacteria:⑧-BL21,⑨-DH5α,⑩-DH5α,⑪-BL21,⑫-BL21

[Huixin Liang][Yueting Guo] [Rui Yao]

II. Agarose gel electrophoresis :⑤⑥⑦ and positive control[Qiyu
Tan][Sirui Dong][Yuxiao Qin]

III. Transformation:⑤⑥⑦ and positive control→BL21[Qiyu Tan][Sirui
Dong][Yuxiao Qin]

IV. Preparation of medium [Huixin Liang][Yueting Guo][Rui Yao]

⑤Input 01 without lac operator-pCOLADuet-1(pCOLADuet-input01)

⑥Input 05 without lac operator-pCOLADuet-1(pCOLADuet-input05)

⑦Input 01 without lac operator-pCDFDuet-1(pCDFDuet-input01)

⑧Input 05 without lac operator-pCDFDuet-1(pCDFDuet-input05)

⑨Input 01 with lac operator-pCOLADuet-1(pCOLADuet-lacO-input01)

⑩Input 05 with lac operator-pCOLADuet-1(pCOLADuet-lacO-input05)

⑪Input 01 with lac operator-pCDFDuet-1(pCDFDuet-lacO-input01)

⑫Input 05 with lac operator-pCDFDuet-1(pCDFDuet-lacO-input05)

Detailed experiments:

I.Shake the bacteria:⑧-BL21,⑨-DH5α,⑩-DH5α,⑪-BL21,⑫-BL21[Huixin
Liang][Yueting Guo] [Rui Yao]

To prepare for the midiprep tomorrow,we shake⑧⑪⑫-BL21(oringinal
tube)and ⑩-DH5α(bacteria conservation from oringinal tube
solution),then store the bacteria and continue to shake overnight.As
we found that we forgot to shake ⑨-DH5α(bacteria conservation from
oringinal tube solution)so we shake it overnight in 15ml tube.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image218.png)

We take the wrong stored bacteria solution in the morning:we did not
take ⑨,take two ⑩(we thought that one is the oringinal tube and one
is stored from the oringial tube) ,so we began to shake ⑨ in the
evening

After shaking for 8h,we found ⑩ (the one from the oringinal
tube)wasn't cloudy,so we suspected it was the antibiotics,so we
aspirated 100μL bacteria solution and added it into another 5ml
LB(without antibiotic) to explore this problem,but after a while we
found that the bacteria solution we thought is from oringinal tube
is actually the stored one,so we discard this tube and continue to
shake the oringinal 2 tubes.

We found only one of the ⑩-DH5α became cloudy in the evening ,so we
only stored its bacteria solution and add 3ml into 200ml LB and
shake overnight

Before shake in conical flask, we stored the bacteria.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image219.png)

Figure

Discription Picture

The bacteria solution we add into 5ml LB to shake

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image220.png)

The group shaken begin at 9:00 a.m.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image221.png)

Shake in conical flask overnight

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image222.png)

II. Agarose gel electrophoresis :⑤⑥⑦ and positive control(Kan+)

[Qiyu Tan][Sirui Dong][Yuxiao Qin]

The concentration of the agarose gel is 1%

Run the gel at 135V for 30min

The loading sequence and system are shown in the following table

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image223.png)

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image224.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image225.png)

III. Transformation:⑤⑥⑦ and positive control→BL21[Qiyu Tan][Sirui
Dong][Yuxiao Qin]

Transformation

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image226.png)

The system (2μL plasmid+50μL competent cells)heat-shocked at 42℃ for
45s,and the other system (4μL plasmid+100μL competent cells)
heat-shocked for 60s

Add 500μL LB medium per tube before shake for 1h and 5min( 220rpm,
37℃)

IV. Shake

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image227.png)

Shake at 37℃,220rpm

When adding 10μL bacterial solution to the PC centrifuge tube
(without antibiotics), add 10μL one more time because it was not
sure whether all 10μL was added, and the actual added volume may be
greater than 10μL.

The tube ⑦-Trans1T1 (with antibiotics) was incorrectly marked and
was discarded and redone.

Before adding bacteria to each tube, it should be mixed upside down.
In this experiment, it was not mixed upside down at first and it's
done after reminding

V. Spread the bacteria solution from transformation on the plates

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image228.png)

Centrifuge all EP tubes were with a micro centrifuge at 1000rpm for
2min, and then pour out part of the supernatant, the remaining
bacterial solution was about 200μL,spread 200μL from transformation
on the plates.

Leave the plates upright for 30min, and invert the plates, incubate
the plates overnight at 37℃

The actual incubator temperature was higher than 37 ° C.

Operator forgot to add LB medium to NC-tube( step12,) so we added
160μL LB before plate coating and then directly coated.

Results of transformation (7.16 observed)

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image229.png)

VI. Preparation of medium [Huixin Liang][Yueting Guo][Rui Yao]

Preparation of LB and plates pouring

Follow protocol:Preparation of LB

We prepared 1L LB medium in total and 400mL of it is solid medium

We pour 14 LB-Str plates in total

Preparation of SOC

Follow protocol: Preparation of SOC medium

We prepared 100ml 1x SOC medium

1. Prepare the medicine, weigh it on a weighing instrument, add it
to a small conical flask and hold it to 100mL

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image230.png)

2.Seal the above LB medium (4 large 2 small):SOC medium (1 small)
and put it into autoclave for sterilization(121℃,20 min)

Discription Picture

Preparation of SOC medium

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image231.png)

Preparation of LB medium

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image232.png)

2024.07.16 Tuesday

Outline:

I.Stored bacteria[Huixin Liang][Yueting Guo][Rui Yao]

II.Maxiprep plasmid and agarose gel electrophoresis:⑧-BL21, ⑩-DH5α,
⑪-BL21, ⑫-BL21 [Huixin Liang] [Yueting Guo] [Rui Yao]

III.Plate streaking:⑤⑥⑦ ,positive control,nuclear actin,nagative
control→BL21[Qiyu Tan][Sirui Dong][Yuetong Ge][Yuxiao Qin]

IV. Preparation of LB medium[Qiyu Tan][Sirui Dong][Yuetong
Ge][Yuxiao Qin]

⑤Input 01 without lac operator-pCOLADuet-1(pCOLADuet-input01)

⑥Input 05 without lac operator-pCOLADuet-1(pCOLADuet-input05)

⑦Input 01 without lac operator-pCDFDuet-1(pCDFDuet-input01)

⑧Input 05 without lac operator-pCDFDuet-1(pCDFDuet-input05)

⑨Input 01 with lac operator-pCOLADuet-1(pCOLADuet-lacO-input01)

⑩Input 05 with lac operator-pCOLADuet-1(pCOLADuet-lacO-input05)

⑪Input 01 with lac operator-pCDFDuet-1(pCDFDuet-lacO-input01)

⑫Input 05 with lac operator-pCDFDuet-1(pCDFDuet-lacO-input05)

Detailed experiments:

I.Stored bacteria [Huixin Liang][Yueting Guo][Rui Yao]

Followed protocol:strain the preservation

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image233.png)

II.Maxiprep plasmid and agarose gel electrophoresis:⑧-BL21, ⑩-DH5α,
⑪-BL21, ⑫-BL21 [Huixin Liang] [Yueting Guo] [Rui Yao]

Maxiprep

Follow protocol:Vigorous plasmid maxiprep

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image234.png)

Agarose gel electrophoresis

Follow protocol:Agarose gel electrophoresis

The concentration of the agarose gel is 1%

Run at 35V for 40min

The load order and system are as follows(unit:μL)

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image235.png)

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image236.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image237.png)

IV. Plate streaking:⑤⑥⑦ ,positive control,nuclear actin,nagative
control→BL21

[Qiyu Tan][Sirui Dong][Yuetong Ge][Yuxiao Qin]

Because it was found that all the shaken bacteria grew(7.15 began
shake,overnight), but none of the coated plates grew(7.15
coat,overnight), which was suspected to be the problem of excessive
incubator/bacterial solution,so we streak the plates to verify our
conjecture.

The groups and results are as follows

Results (7.17 observed)

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image238.png)

IV. Preparation of LB medium[Qiyu Tan][Sirui Dong][Yuetong
Ge][Yuxiao Qin]

Followed protocol:LB liquid medium

We prepare 1.6L LB in total

We pour 16 plates with Kan and 16 plates with Stc

2024.07.17 Wednesday

Outline:

I.Antibiotic resistance verification:Kan,Str[Qiyu Tan][Sirui
Dong][Yuetong Ge]

II.Transformation:nuclear actin/⑨/⑩→BL21/transB/transetta

[Qiyu Tan][Sirui Dong][Yuetong Ge][Yuxiao Qin]

III.Without/double digestion and agarose gel electrophoresis:⑥⑦⑧⑨⑩,
nuclear actin(positive control)[Huixin Liang][Yueting Guo][Rui Yao]

IV.Overnight digestion:⑦⑧⑩[Huixin Liang][Yueting Guo][Rui Yao]

V.Shake the bacteria:⑤-BL21, ⑨-BL21[Qiyu Tan][Sirui Dong][Yuetong
Ge]

VI.Preparation of LB medium[Qiyu Tan][Sirui Dong][Yuetong Ge]

⑤Input 01 without lac operator-pCOLADuet-1(pCOLADuet-input01)

⑥Input 05 without lac operator-pCOLADuet-1(pCOLADuet-input05)

⑦Input 01 without lac operator-pCDFDuet-1(pCDFDuet-input01)

⑧Input 05 without lac operator-pCDFDuet-1(pCDFDuet-input05)

⑨Input 01 with lac operator-pCOLADuet-1(pCOLADuet-lacO-input01)

⑩Input 05 with lac operator-pCOLADuet-1(pCOLADuet-lacO-input05)

⑪Input 01 with lac operator-pCDFDuet-1(pCDFDuet-lacO-input01)

⑫Input 05 with lac operator-pCDFDuet-1(pCDFDuet-lacO-input05)

Detailed experiments:

I.Antibiotic resistance verification:Kan,Str[Qiyu Tan][Sirui
Dong][Yuetong Ge][Yuxiao Qin]

Due to the growth of nagative control,we suspect that the antibiotic
may not suffecient,so we verified the resistance of antibiotic in
several ways.

We use BL21(DE3) without transformation to streak/coat the plates
and shake the bacteria to verify the resistance of Kan and Str

Plates streaking and coating

The bacteria solution used in verification is made by mixing 90μL LB
medium and 10μL BL21(DE3) competent cells

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image239.png)

Results (7.17 observed)

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image240.png)

Bacteria shaking

We use three kinds of competent cells (BL21, TransB, and Transetta)
to verify the resistance of three antibiotics (Kan5.21/Kan11.30/Str)

Kan(11.30) and Str are the antibiotic we used,Kan(11.30) is borrowed
from others to set up a positive control

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image241.png)

Concentration of antibiotic:

Kan(5.30):200mg/ml

Kan(11.30):100mg/ml

Str:200mg/ml

Results (7.18 observed)

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image242.png)

Analysis

Our antibiotic(Kan 5.21 and Str) are not suffecient

V. Transformation:nuclearactin/⑨/⑩→BL21/transB/transetta

[Qiyu Tan][Sirui Dong][Yuetong Ge][Yuxiao Qin]

Follow protocol:Bacterial Transformation

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image243.png)

After add plasmids into competent cells and on ice for 30min,we
found that we forgot to turn on the water bath pot,so we continue to
place the mixture on ice for 11min until the pot was ready.

Heat-shock at 42℃for 1min

Add 500μL SOC medium per tube before shake for 1h( 220rpm, 37℃)

Spread 200μL bacteria solution from transformation on the plate

Place the coated plate at 37℃ for 30min. After the bacterial
solution dried, invert and cultivate overnight.

Moisturize all plates with wet filter paper and sponge

Results(7.18 observed)

Since it was confirmed that antibiotic resistance was problematic,
all plates were discarded and re-tested

VI. Without/double digestion and agarose gel electrophoresis
:⑥⑦⑧⑨⑩,nuclear actin(positive control)

[Huixin Liang][Yueting Guo][Rui Yao]

Follow protocol:Restriction Digest of Plasmid DNA Agarose gel
electrophoresis

Double digestion

The system are as follows:

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image244.png)

Digest at 37℃ for 30min

Agarose gel electrophoresis

The concentration of the agarose gel is 1%

Run the gel at 135V for 40 min

The loading sequence and system are shown in the following table

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image245.png)

unit:μL

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image246.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image247.png)

Analysis

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image248.png)

IV.Overnight digestion:⑦⑧⑩[Huixin Liang][Yueting Guo][Rui Yao]

According to the results of the gel, we dicided to perform overnight
enzyme digestion on ⑦⑧⑩ plasmids

Follow peotocol:Restriction Digest of Plasmid DNA

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image249.png)

Digest at roomtemperature overnight

V. Shake the bacteria:⑤-BL21, ⑨-BL21[Qiyu Tan][Sirui Dong][Yuetong
Ge]

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image250.png)

Shake at 220rpm,37℃

The remaining post-shake bacterial solution in the 15-ml tube was
stored in a 4-degree refrigerator

Results

Discard all bacteria tomorrow because of the abnormal phenomenon

Figure

Discription Picture

Shake the bacteria

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image251.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image252.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image253.png)

VI.Preparation of LB medium [Qiyu Tan][Sirui Dong][Yuetong Ge]

Follow protocol: Preparation of LB

Prepare 1.6L in total

Add the solid ingredients suitable for the 1.5L system, and adjust
to 1.6L due to constant volume error

2024.07.18 Thursday

Outline:

I.Agarose gel electrophoresis:⑦⑧⑩[Huixin Liang][Yueting Guo][Rui
Yao]

II.Single/double digestion and ararose gel
eletrophoresis:⑦⑧⑪⑫[Huixin Liang][Yueting Guo][Rui Yao]

III.Shake the bacteria:⑤-BL21, ⑨-BL21 and store[Huixin
Liang][Yueting Guo][Rui Yao]

IV.Plate coating:nuclear actin/⑨/⑩→BL21/transB/transetta[Yuxiao
Qin][Sirui Dong][Qiyu Tan][Yuetong Ge]

V.Preparation of preparing competent cells:⑦-BL21[Huixin
Liang][Yuetong Ge]

VI.Antibiotic resistance verification[Huixin Liang][Yuetong Ge]

⑤Input 01 without lac operator-pCOLADuet-1(pCOLADuet-input01)

⑥Input 05 without lac operator-pCOLADuet-1(pCOLADuet-input05)

⑦Input 01 without lac operator-pCDFDuet-1(pCDFDuet-input01)

⑧Input 05 without lac operator-pCDFDuet-1(pCDFDuet-input05)

⑨Input 01 with lac operator-pCOLADuet-1(pCOLADuet-lacO-input01)

⑩Input 05 with lac operator-pCOLADuet-1(pCOLADuet-lacO-input05)

⑪Input 01 with lac operator-pCDFDuet-1(pCDFDuet-lacO-input01)

⑫Input 05 with lac operator-pCDFDuet-1(pCDFDuet-lacO-input05)

Detailed experiments:

I. Agarose gel electrophoresis:⑦⑧⑩[Huixin Liang][Yueting Guo][Rui
Yao]

The concentration of the agarose gel is 1%

Run the gel at 135V for 40min

The loading sequence and system are shown in the following table

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image254.png)

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image255.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image256.png)

II.Single/double digestion and ararose gel nucleic acid
eletrophoresis:⑦⑧⑪⑫[Huixin Liang] [Yueting Guo] [Rui Yao]

Digest at 37℃ for 30min

The concentration of the agarose gel is 1%

Run the gel at 135V for 40 min

The digestion system and the loading sequence and system are shown
in the following table

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image257.png)

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image258.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image259.png)

Analysis

⑦ ⑧ ⑪ ⑫ The preliminary analysis of the enzyme band is correct, but
there is a problem that the band is trailing

The solidification time is too short after the gel is dispensed, and
the formation of the gap is too shallow. Next time, we should
prolong the solidification time appropriately.

The temperature of the process of running the gel is too high. Pay
attention to use more ice boxes to cool down the temperature, and
observe the situation frequently.

III.Shake the bacteria:⑤-BL21, ⑨-BL21 [Huixin Liang][Yueting
Guo][Rui Yao]

We shake the bacteria for maxi plasmid prep tomorrow

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image260.png)

Figure

Discription Picture

Shake in conical flask

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image261.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image262.png)

IV.Plate coating:nuclear actin/⑨/⑩→BL21/transB/transetta[Yuxiao
Qin][Sirui Dong][Qiyu Tan][Yuetong Ge]

Since the plate we used before was not resistant, we re-poured the
plates with effective resistance and spread the bacteria solution
from transformation in 7.17 on new plates.

We pour plates twice,a total of 25 plates with Kan and 5 plates with
Str.

We coat plates as following table

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image263.png)

Results (7.19 observed)

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image264.png)

V.Preparation of preparing competent cells:⑦-BL21[Huixin
Liang][Yuetong Ge]

Follow protocol:The preparation before preparing competent cells

We prepare materials for the preparation of competent cells tomorrow

The steps of preparation are as follows

1. Prepare strain: ⑦Input 01 without lac operator-pCDFDuet-1 (Str)

2. Preparation of medicines and lance tips

Control chemical: anhydrous calcium chloride, glycerol (glycerol)

3. Weigh the drug: weigh 0.882g CaCl2

a) Take a quarter of the weighing paper and place it in an
electronic balance (Precise to four decimal places) and fold it
twice

b) Use the blue pipette tip to pick the granular anhydrous calcium
chloride and place it in a EP tube on the EP tube rack for weighing

c) Theoretical weight:0.882g ,Actual weight: 0.9001g

4. Preparation of CaCl2/glycerol 100mL:

add 0.9001g CaCl2 and 10mL glycerol ( cut off the tip of the tip of
the pipette to suck), and use a 50mL centrifuge tube to take RO
water in two times, seal the mouth and mix.

5. autoclave:

a) A flask of non-resistant LB (200mL);

b) Sterilisation CaCl2/glycerol 100mL (4 configurations) Pay
attention to aeration;.

c) Sterilise 15mL and 50mL centrifuge tubes in one box, and
sterilise 1.5mL EP tubes (more than 50 in two boxes).

d) Sterilise one box of blue and yellow pipette tip each,
andsterilise half box of 5mL large pipette tip. Packed them in kraft
paper.

VI.Antibiotic resistance verification[Huixin Liang][Yuetong Ge]

We verify resistance by putting 3 kinds of different bacteria into 2
kinds of resistance (stc, Kan) and non-resistant LB medium

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image265.png)

Analysis

1. Kan and Stc are suffecient

2. Plasmid⑦ has spectomycin resistance genes

Figure

Discription Picture

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image266.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image267.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image268.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image269.png)

2024.07.19 Friday

Outline:

I.Maxiprep,digestion and agarise gel electrophoresis:⑤-BL21, ⑨-BL21
[Huixin Liang][Yueting Guo][RuiYao][YuxiaoQin]

II.Preparation of competent cells:⑦-BL21[Huixin Liang][Yuetong Ge]

III.Transformation:nuclear actin/⑨/⑩→BL21/transB/transetta [Qiyu
Tan][Sirui Dong

③→⑦-BL21/trans1t1[Huixin Liang][Yuetong Ge]

IV.Preparation of preparing competent cells:⑧-BL21,⑪-BL21,⑫-BL21
[Huixin Liang][Yuetong Ge]

V.Pour plates [Huixin Liang][Yuetong Ge]

③H01 without lac operator-pET-15b(pET-15b-H01-linker-EGFP)

⑤Input 01 without lac operator-pCOLADuet-1(pCOLADuet-input01)

⑦Input 01 without lac operator-pCDFDuet-1(pCDFDuet-input01)

⑨Input 01 with lac operator-pCOLADuet-1(pCOLADuet-lacO-input01)

⑩Input 05 with lac operator-pCOLADuet-1(pCOLADuet-lacO-input05)

⑪Input 01 with lac operator-pCDFDuet-1(pCDFDuet-lacO-input01)

⑫Input 05 with lac operator-pCDFDuet-1(pCDFDuet-lacO-input05)

Detailed experiments:

I.Maxiprep,digestion and agarise gel electrophoresis:⑤-BL21,
⑨-BL21[Huixin Liang][Yueting Guo][RuiYao][YuxiaoQin]

To make sure the bacteria the company gave us contained the right
plasmid,we prep plasmids(shook in 7.18) by two methods(EndoFree
MaxiPlasmid Kit & Vigorous plasmid maxprep kit ) from them and run
agarose gel to verify.

Follow protocol:Maxiprep ,digestion and agarose gel electrophoresis

Results of maxiprep(EndoFree MaxiPlasmid Kit)

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image270.png)

units:ng/μL

Results of maxiprep(Vigorous plasmid maxprep kit )

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image271.png)

Digest at 37℃ for 30min

The concentration of the agarose gel is 1%

Run the gel at 135V for 40 min

The digestion system and the loading sequence and system are shown
in the following table

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image272.png)

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image273.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image274.png)

Figure

Discription Picture

Maxiprep

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image275.png)

Get the plasmid

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image276.png)

II.Preparation of competent cells:⑦-BL21[Huixin Liang][Yuetong Ge]

1. Put 200ml bacterial solution into four 50ml tubes (sterilized and
pre-cooled), seal with a sealing film. Then ssstand on ice for 10
minutes, and mark the 50ml tubes

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image277.png)

The bacterial solution used in this step should be slightly cloudy,
not too cloudy or too clear, specifically refer to the following
figure:

2.1600g, centrifuge at 4℃ for 10min, note that the centrifuge should
be refrigerated in advance

3. Melt glycerin /CaCl2 (mixture) in a hot water separator

4. Discard the supernatant, add 10ml glycerin /CaCl2 to each bottle,
pump and mix well, and seal with a sealing film

5.2470rpm, 1100g centrifuge for 5min

6.2 tubes were re-centrifuged due to precipitation suspension, and
the other two tubes discarded the supernatant, added 10ml glycerol
/CaCl2 and blew several times. The two tubes re-centrifuged were
subjected to the same operation after centrifugation

7. Ice bath for 30min

8. Centrifuge 1100g again for 5min and discard the supernatant

9. Add 1ml glycerin /CaCl2 to each tube to dissolve the
precipitation, then add 1mL glycerin /CaCl2, suction and mix well

10.Pick up 50 EP tubes with tweezers, mark "7" on the lid, add
50-100μL bacterial solution to each tube, and store them in the
refrigerator at -80℃

III.Transformation:nuclear actin/⑨/⑩→BL21/transB/transetta[Qiyu
Tan][Sirui Dong]

③→⑦-BL21/trans1t1[Huixin Liang][Yuetong Ge]

As we failed in transformation for many times,Tan and Dong decide to
learn new steps of transformation from Ms.Qian,they transform
plasmid⑨⑩ and nuclear actin(positive control) to 3 kinds of
competent cells: BL21,TransB and transetta.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image278.png)

Heat-shock at 42℃for 90s and on ice for 1min

Add 500μL LB medium per tube before shake for 1h( 220rpm, 37℃)

Spread 200μL bacteria solution from transformation on the plate

Spread the solution from transformation on the plates until the
bacteria solution dried,then Invert the coated plate at 37℃ and
cultivate overnight.

To test the function of LIRA,Liang and Ge transform ③ into
⑦-BL21(Just made into competent cells)

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image279.png)

Heat-shock at 42℃for 45s and on ice for 2min

Add 500μL SOC medium per tube before shaking for 1h( 220rpm, 37℃)

Spread 200μL bacteria solution from transformation on the plate

Place the coated plate at 37℃ for 30min. After the bacterial
solution dried, invert and cultivate overnight.

Results (7.20 observed)

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image280.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image281.png)

IV.Preparation of preparing competent
cells:⑧-BL21,⑪-BL21,⑫-BL21[Huixin Liang][Yuetong Ge]

Follow protocol: The preparation of preparing competent cells

V.Pour plates[Huixin Liang][Yuetong Ge]

We pour 15 plates in total,5 with Stc,5 with Amp and 5 with Amp+Stc

The concentration of antibiotic is 100μg/ml

2024.07.20

Outline:

I.Maxiprep for plasmids(Tiangen and Vigorous):⑦-BL21[Yueting
Guo][Rui Yao][Yuxiao Qin]

II.Single/double digestion:⑦⑨[Yueting Guo][Rui Yao][Yuxiao Qin]

III.Shake bacteria:③→⑦-BL21 [Huixin Liang][Yuetong Ge]

IV.Preparation of making competent cells:⑤-BL21, ⑨-BL21[Huixin
Liang][Yuetong Ge]

V.Transformation:③→⑦-BL21/BL21/trans1t1[Huixin Liang][Yuetong Ge]

②+⑩/④+⑥/④+nuclear actin→BL21/transB/transetta[Qiyu Tan][Sirui Dong]

VI.Preparation of competent cells:⑧-BL21,⑪-BL21,⑫-BL21[Huixin
Liang][Yuetong Ge]

VII.Preparation of LB medium and plates pouring[Qiyu Tan][Sirui
Dong]

②H05 with lac operator-pET-15b(pET-15b-lacO-H05-linker-EGFP)

③H01 without lac operator-pET-15b(pET-15b-H01-linker-EGFP)

④H05 without lac operator-pET-15b(pET-15b-H05-linker-EGFP)

⑤Input 01 without lac operator-pCOLADuet-1(pCOLADuet-input01)

⑥Input 05 without lac operator-pCOLADuet-1(pCOLADuet-input05)

⑦Input 01 without lac operator-pCDFDuet-1(pCDFDuet-input01)

⑧Input 05 without lac operator-pCDFDuet-1(pCDFDuet-input05)

⑨Input 01 with lac operator-pCOLADuet-1(pCOLADuet-lacO-input01)

⑩Input 05 with lac operator-pCOLADuet-1(pCOLADuet-lacO-input05)

⑪Input 01 with lac operator-pCDFDuet-1(pCDFDuet-lacO-input01)

⑫Input 05 with lac operator-pCDFDuet-1(pCDFDuet-lacO-input05)

Detailed Experiments:

I.Maxiprep for plasmids(Tiangen and Vigorous):⑦-BL21[Yueting
Guo][Rui Yao][Yuxiao Qin]

Follow protocol:Maxiprep

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image282.png)

II. Single/double digestion:⑦⑨[Yueting Guo][Rui Yao][Yuxiao Qin]

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image283.png)

Digest at room temperature overnight

III. Shake bacteria:③→⑦-BL21 [Huixin Liang][Yuetong Ge]

We Pick colony of ⑦-BL21 that successfully transformed plasmid ③
from the double-resistant (Amp+stc)plate and shake

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image284.png)

IV.Preparation of Making competent cells:⑤-BL21, ⑨-BL21[Huixin
Liang][Yuetong Ge]

Follow protocol:The preparation of making competent cells

V.Transformation:③→⑦-BL21/BL21/trans1t1[Huixin Liang][Yuetong Ge]

Follow protocol:Transformation

Heat-shock at 42℃for 90s and on ice for 2min

Add 500μL SOC medium per tube before shake for 1h( 220rpm, 37℃)

Spread 200μL bacteria solution from transformation on the plate

Place the coated plate at 37℃ for 30min. After the bacterial
solution dried, invert and cultivate overnight.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image285.png)

Results(7.21 observed)

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image286.png)

Figure

Discription Picture

Results of transformation

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image287.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image288.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image289.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image290.png)

②+⑩/④+⑥/④+nuclear actin→BL21/transB/transetta[Qiyu Tan][Sirui Dong]

l Heat-shock at 42℃for 95s and on ice for 1min

Add 500μL LB medium per tube before shake for 1h( 220rpm, 37℃)

Spread 200μL bacteria solution from transformation on the plate

Place the coated plate at 37℃ for 30min. After the bacterial
solution dried, invert and cultivate overnight.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image291.png)

Results(7.21 observed)

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image292.png)

VI.Preparation of competent cells:⑧-BL21,⑪-BL21,⑫-BL21[Huixin
Liang][Yuetong Ge]

Follow protocol:The preparation of competent cells

VII.Preparation of LB medium and plates pouring[Qiyu Tan][Sirui
Dong]

Follow protocol:The preparation of LB medium

We prepare 4L in total,and 2L of them was used to pour plates

We pour 3 types of plates:withAmp,withKan,withAmp+Kan

We pour 9 plates of each type

2024.07.21 Sunday

Outline:

I.Agarose gel electrophoresis:⑦⑨[Yueting Guo][Yuxiao Qin]

II.Shake bacteria:②+⑩/④+⑥/④+Nuclear
actin→BL21/transB/transetta(transformed successfully),store and
shake ⑥-DH5α[Qiyu Tan][Sirui Dong]

③→⑦-BL21 and store [Yuetong Ge][Rui Yao][Yueting Guo]

III.Preparation of competent cells:⑤-BL21, ⑨-BL21[Yuetong Ge][Rui
Yao]

②H05 with lac operator-pET-15b(pET-15b-lacO-H05-linker-EGFP)

③H01 without lac operator-pET-15b(pET-15b-H01-linker-EGFP)

④H05 without lac operator-pET-15b(pET-15b-H05-linker-EGFP)

⑤Input 01 without lac operator-pCOLADuet-1(pCOLADuet-input01)

⑥Input 05 without lac operator-pCOLADuet-1(pCOLADuet-input05)

⑦Input 01 without lac operator-pCDFDuet-1(pCDFDuet-input01)

⑧Input 05 without lac operator-pCDFDuet-1(pCDFDuet-input05)

⑨Input 01 with lac operator-pCOLADuet-1(pCOLADuet-lacO-input01)

⑩Input 05 with lac operator-pCOLADuet-1(pCOLADuet-lacO-input05)

⑪Input 01 with lac operator-pCDFDuet-1(pCDFDuet-lacO-input01)

⑫Input 05 with lac operator-pCDFDuet-1(pCDFDuet-lacO-input05)

Detailed Experiments:

I.Agarose gel electrophoresis:⑦⑨[Yueting Guo][Yuxiao Qin]

To ensure the plasmids contained in bacteria are correct,we
extracted plasmid⑦ by EndoFree MaxiPlasmid Kit & Vigorous plasmid
maxprep kit and ⑨ by Vigorous plasmid maxprep kit,then digest them
by enzyme at room temperature overnight in 7.20,and run agarose gel
today.

Follow protocol:The agarose gel electrophoresis

The concentration of the agarose gel is 1%

Run the gel at 135V for 40 min

The loading sequence and system are shown in the following table

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image293.png)

unit:μL

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image294.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image295.png)

II.Shake bacteria:②+⑩/④+⑥/④+nuclear
actin→BL21/transB/transetta(transformed successfully)[Qiyu
Tan][Sirui Dong]

We pick single colonies from the plates that grew normally,and
recoat the abnormal growth:cotransformation ④+⑥→BL21 K+A+ plate

Shake at 36.5℃,250rpm

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image296.png)

Store and shake ⑥-DH5α[Qiyu Tan][Sirui Dong]

Because plasmid⑥ was going to run out,so we decided to prep some for
further experiments.We shook the ⑥-DH5α in 15ml tube overnight in
7.20,and store the bacteria in 7.21,then we shake it in conical
flask overnight to prepare for the maxiprep tomorrow

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image297.png)

Shake at 37℃,250rpm

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image298.png)

Pick single colonies:③→⑦-BL21[Yuetong Ge][Rui Yao][Yueting Guo]

We pick single colonies from ③→⑦-BL21 A+S+ plate and shake bacteria
for miniprep tomorrow

Shake at 37℃,250rpm

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image299.png)

Store bacteria:③→⑦-BL21[Yuetong Ge][Rui Yao][Yueting Guo]

After shaking for 8.5h,we store bacteria and store the rest of
bacteria solution in 15ml tube at 4℃

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image300.png)

III.Preparation of competent cells:⑤-BL21, ⑨-BL21[Yuetong Ge][Rui
Yao]

Follow protocol: The preparation of competent cells

Monday/2024.07.22

Outline:

I. Miniprep/Maxiprep :③→⑦-BL21(miniprep) [Yueting Guo][Yuetong Ge]

②+⑩→BL21,④+⑥→transB,④+nuclear actin→BL21(miniprep)[Sirui Dong]

⑥-DH5α(maxiprep)[Qiyu Tan][Rui Yao][Yuxiao Qin]

II.Digestion:③→⑦-BL21 [Yueting Guo][Yuetong Ge]

⑥-DH5α[Qiyu Tan][Rui Yao][Yuxiao Qin]

②+⑩→BL21,④+⑥→transB,④+nuclear actin→BL21[Sirui Dong]

III.Transformation:④→⑧-BL21,①→⑪-BL21,②→⑫-BL21,③→⑤-BL21,①→⑨-BL21[Yueting
Guo][Yuetong Ge]

②H05 with lac operator-pET-15b(pET-15b-lacO-H05-linker-EGFP)

③H01 without lac operator-pET-15b(pET-15b-H01-linker-EGFP)

④H05 without lac operator-pET-15b(pET-15b-H05-linker-EGFP)

⑤Input 01 without lac operator-pCOLADuet-1(pCOLADuet-input01)

⑥Input 05 without lac operator-pCOLADuet-1(pCOLADuet-input05)

⑦Input 01 without lac operator-pCDFDuet-1(pCDFDuet-input01)

⑧Input 05 without lac operator-pCDFDuet-1(pCDFDuet-input05)

⑨Input 01 with lac operator-pCOLADuet-1(pCOLADuet-lacO-input01)

⑩Input 05 with lac operator-pCOLADuet-1(pCOLADuet-lacO-input05)

⑪Input 01 with lac operator-pCDFDuet-1(pCDFDuet-lacO-input01)

⑫Input 05 with lac operator-pCDFDuet-1(pCDFDuet-lacO-input05)

Detailed Experiments:

I.Miniprep/Maxiprep :③→⑦-BL21(miniprep) [Yueting Guo][Yuetong Ge]

②+⑩→BL21,④+⑥→transB,④+nuclear actin→BL21(miniprep)[Sirui Dong]

⑥-DH5α(maxiprep)[Qiyu Tan][Rui Yao][Yuxiao Qin]

Followed protocol:Miniprep and maxiprep

Results of ③→⑦-BL21(miniprep)

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image301.png)

Results of ②+⑩→BL21,④+⑥→transB,④+nuclear actin→BL21(miniprep)

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image302.png)

Results of ⑥-DH5α(maxiprep)

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image303.png)

II.Digestion:③→⑦-BL21 [Yueting Guo][Yuetong Ge]

②+⑩→BL21,④+⑥→transB,④+nuclear actin→BL21[Sirui Dong]

⑥-DH5α[Qiyu Tan][Rui Yao][Yuxiao Qin]

Follow protocol: Digestion

Digestion:③→⑦-BL21

The system are as follows

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image304.png)

Digest at room temperature overnight

DIgestion:②+⑩→BL21,④+⑥→transB,④+nuclear actin→BL21

The system are as follows

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image305.png)

Digest at room temperature overnight

Digestion:⑥-DH5α

The system are as follows

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image306.png)

Digest at room temperature overnight

III.Transformation:④→⑧-BL21,①→⑪-BL21,②→⑫-BL21,③→⑤-BL21,①→⑨-BL21[Yueting
Guo][Yuetong Ge]

Foolow protocol:Transformation

⑧⑪⑫⑤⑨-BL21 have been made into conpetent cells

The plasmid was first diluted to 250ng/μL

2μL of the corresponding plasmid was added to 50μL of the competent
cells,

Heat shocked at 42℃ for 90s, and on ice for 2min.

Add 500μL of SOC, and then placed obliquely on a shaker at 180rpm
for 1h+.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image307.png)

Results (7.23 observed)

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image308.png)

2024.07.23 Tuesday

Outline:

I.Agarose gel electrophoresis:③→⑦-BL21,②+⑩→BL21,④+⑥→transB,④+nuclear
actin→BL21(mini),⑥-DH5α[Qiyu Tan][Rui Yao][Yuxiao Qin]

II.Shake the bacteria:③→⑦-BL21,②+⑩→BL21,④+⑥→transB,④+nuclear
actin→BL21

[Qiyu Tan][Rui Yao][Yuxiao Qin][Yueting Guo][Yuetong Ge]

III.Transformation:③→⑦-BL21/BL21,④→⑧-BL21/BL21,①→⑪-BL21/BL21,②→⑫-BL21/BL21,③→⑤-BL21/BL21,①→⑨-BL21/BL21
[Qiyu Tan][Yuetong Ge]

IV.Preparation of LB medium and plate pouring

V.Plasmid extraction(mini prep):④+⑥→BL21

②H05 with lac operator-pET-15b(pET-15b-lacO-H05-linker-EGFP)

③H01 without lac operator-pET-15b(pET-15b-H01-linker-EGFP)

④H05 without lac operator-pET-15b(pET-15b-H05-linker-EGFP)

⑤Input 01 without lac operator-pCOLADuet-1(pCOLADuet-input01)

⑥Input 05 without lac operator-pCOLADuet-1(pCOLADuet-input05)

⑦Input 01 without lac operator-pCDFDuet-1(pCDFDuet-input01)

⑧Input 05 without lac operator-pCDFDuet-1(pCDFDuet-input05)

⑨Input 01 with lac operator-pCOLADuet-1(pCOLADuet-lacO-input01)

⑩Input 05 with lac operator-pCOLADuet-1(pCOLADuet-lacO-input05)

⑪Input 01 with lac operator-pCDFDuet-1(pCDFDuet-lacO-input01)

⑫Input 05 with lac operator-pCDFDuet-1(pCDFDuet-lacO-input05)

Detailed Experiments:

I. Agarose gel electrophoresis:plasmid preped from

③→⑦-BL21,②+⑩→BL21,④+⑥→transB,④+nuclear actin→BL21(mini),⑥-DH5α[Qiyu
Tan][Rui Yao][Yuxiao Qin]

Follow protocol:Agarose gel electrophoresis

plasmid ⑥ was maxipreped from the original bacteria,single and
double digested overnight

plasmid ②+⑩, NA+④,④+⑥ were minipreped from the transformed bacteria

Use 1% agarose gel

Run at 135V for 40min

The system is as follows(unit:μL):

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image309.png)

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image310.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image311.png)

unit:μL

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image312.png)

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image313.png)

Analysis

1. ⑥All digestion showed nearly correct banding, including single
and double digestion, without any impurity

2. ⑥No digestion showed three bands, and there may be linear,
annular, or superhelix structure.

3. No bands at all in co-transformation, but no impurity bands
either. It is speculated that this is a small extraction problem,
and the measured concentration is also relatively low,would do big
extraction next

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image314.png)

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image315.png)

Analysis

We couldn't see any bands on the gel,which we thought no plasmid has
been extracted.

We decided to shake the bacteria again in order to do Vigrous
plasmid extraction.

II.Shake the bacteria:③→⑦-BL21,②+⑩→BL21,④+⑥→transB,④+nuclear
actin→BL21

[Qiyu Tan][Rui Yao][Yuxiao Qin][Yueting Guo][Yuetong Ge]

Because the electrophoretic bands of mini plasmid extraction after
the above transformation were incorrect, take the corresponding
bacterial solution and expand in 200mL LB,waiting for Maxiprep
tomorrow

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image316.png)

II.Transformation:③→⑦-BL21/BL21,④→⑧-BL21/BL21,①→⑪-BL21/BL21,②→⑫-BL21/BL21,③→⑤-BL21/BL21,①→⑨-BL21/BL21
[Qiyu Tan][Yuetong Ge]

Follow protocol:Transformation

Heat-shock at 42℃for 90s and on ice for 2min

Add 500μL SOC medium per tube before shake for 1h( 220rpm, 37℃)

Spread 200μL bacteria solution from transformation on the plate

Invert the coated plate overnight at 37℃

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image317.png)

Results (7.24 observed)

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image318.png)

Figure

Discription Picture

Results of transformation

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image319.png)

III.Preparation of LB medium and plate pouring

Follow protocol:Preparation of LB medium

Prepare 2 groups in total, 1L of the first group has been poured
into the plate, and 2.4L of the second group)

2024.07.24 Wednesday

Outline:

I.Maxiprep and digestion:③→⑦-BL21,②+⑩→BL21,④+⑥→BL21,④→nuclear
actin-BL21[Qiyu Tan][Rui Yao][Yuxiao Qin]

II.Transformation:③→⑦-BL21/BL21,①→⑪-BL21/BL21,②→⑫-BL21/BL21,③→⑤-BL21/BL21,①→⑨-BL21/BL21[Huixin
Liang][Yuetong Ge][Saiyu Luo]

III.Pick single colony and shake
bacteria:④→⑧-BL21,①→⑨-BL21,③→⑤-BL21[Huixin Liang][Yuetong Ge][Saiyu
Luo]

IV.Preparation of LB medium [Qiyu Tan][Rui Yao][Yuxiao Qin]

①H01 with lac operator-pET-15b(pET-15b-lacO-H01-linker-EGFP)

②H05 with lac operator-pET-15b(pET-15b-lacO-H05-linker-EGFP)

③H01 without lac operator-pET-15b(pET-15b-H01-linker-EGFP)

⑤Input 01 without lac operator-pCOLADuet-1(pCOLADuet-input01)

⑥Input 05 without lac operator-pCOLADuet-1(pCOLADuet-input05)

⑦Input 01 without lac operator-pCDFDuet-1(pCDFDuet-input01)

⑧Input 05 without lac operator-pCDFDuet-1(pCDFDuet-input05)

⑨Input 01 with lac operator-pCOLADuet-1(pCOLADuet-lacO-input01)

⑩Input 05 with lac operator-pCOLADuet-1(pCOLADuet-lacO-input05)

⑪Input 01 with lac operator-pCDFDuet-1(pCDFDuet-lacO-input01)

⑫Input 05 with lac operator-pCDFDuet-1(pCDFDuet-lacO-input05)

Detailed Experiments:

I.Maxiprep and digestion:③→⑦-BL21,②+⑩→BL21,④+⑥→BL21,④→nuclear
actin-BL21[Qiyu Tan][Rui Yao][Yuxiao Qin]

Follow protocol:Maxiprep

Since the precipitate was not yet completely separated from the
supernatant, repeat the centrifugation after adding 5mL BufferIII,
on ice for 12min and centrifugation

Step 6 mistakenly added 5mL Buffer I, so centrifuge for two minutes,
add 10mL isopropyl alcohol again, and repeat the operation after
adding isopropyl alcohol

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image320.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image321.png)

Digest overnigt at room temperature

II.Transformation:③→⑦-BL21/BL21,①→⑪-BL21/BL21,②→⑫-BL21/BL21,③→⑤-BL21/BL21,①→⑨-BL21/BL21
[Huixin Liang][Yuetong Ge][Saiyu Luo]

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image322.png)

results (7.25 observed)

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image323.png)

Figure

Discription Picture

Result of transformation

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image324.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image325.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image326.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image327.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image328.png)

III. Shake bacteria:④→⑧-BL21,①→⑨-BL21,③→⑤-BL21 and store[Huixin
Liang][Yuetong Ge][Saiyu Luo]

Pick the single colonies from successfuly transformed plates (double
resistances)and shake at 220rpm,37℃

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image329.png)

Store the bacteria

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image330.png)

Figure

Discription Picture

Store the bacteria

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image331.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image332.png)

IV.Preparation of LB medium [Qiyu Tan][Rui Yao][Yuxiao Qin]

Follow protocol:Preparation of LB medium

Prepare 2L LB in total

2024.07.25 Thursday

Outline:

I.Agarose gel electrophoresis:③→⑦-BL21,②+⑩→BL21,④+⑥→BL21,④+nuclear
actin→BL21[Qiyu Tan][Sirui Dong][Rui Yao][Yuxiao Qin]

II.Single/double digestion:③→⑦-BL21,②+⑩→BL21,④+⑥→BL21 [Qiyu
Tan][Sirui Dong][Rui Yao][Yuxiao Qin]

III.IPTG induction:③→⑦-BL21,①→⑨-BL21 [Huixin Liang][Yuetong Ge]

IV.PIck single colony and shake : ③→⑦-BL21, ①→⑪-BL21, ③→⑤-BL21,
①→⑨-BL21 [Huixin Liang][Yuetong Ge][Qiyu Tan]

V.Store the bacteria : ③→⑦-BL21(7.20), ①→⑨-BL21(7.23),
④→⑧-BL21(7.23), ③→⑤-BL21(7.23), ③→⑦-BL21(7.25), ①→⑪-BL21(7.25),
③→⑤-BL21(7.25), ①→⑨-BL21(7.25)[Huixin Liang][Yuetong Ge][Qiyu Tan]

VI.Transformation:②→⑫-BL21/BL21[Huixin Liang][Yuetong Ge][Qiyu Tan]

①H01 with lac operator-pET-15b(pET-15b-lacO-H01-linker-EGFP)

②H05 with lac operator-pET-15b(pET-15b-lacO-H05-linker-EGFP)

③H01 without lac operator-pET-15b(pET-15b-H01-linker-EGFP)

④H05 without lac operator-pET-15b(pET-15b-H05-linker-EGFP)

⑤Input 01 without lac operator-pCOLADuet-1(pCOLADuet-input01)

⑥Input 05 without lac operator-pCOLADuet-1(pCOLADuet-input05)

⑦Input 01 without lac operator-pCDFDuet-1(pCDFDuet-input01)

⑧Input 05 without lac operator-pCDFDuet-1(pCDFDuet-input05)

⑨Input 01 with lac operator-pCOLADuet-1(pCOLADuet-lacO-input01)

⑩Input 05 with lac operator-pCOLADuet-1(pCOLADuet-lacO-input05)

⑪Input 01 with lac operator-pCDFDuet-1(pCDFDuet-lacO-input01)

⑫Input 05 with lac operator-pCDFDuet-1(pCDFDuet-lacO-input05)

Detailed Experiments:

I.Agarose gel electrophoresis:③→⑦-BL21,②+⑩→BL21,④+⑥→BL21,④+nuclear
actin→BL21[Qiyu Tan][Sirui Dong][Rui Yao][Yuxiao Qin]

Agar-gel electrophoresis: Four groups of Maxiprep plasmids (digested
overnight at 37℃ yesterday)

The loading order and system are as follows(unit:μL)

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image333.png)

-0 means it's not digested by enzyme,-2 means it's digested by
double enzyme

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image334.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image335.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image336.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image337.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image338.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image339.png)

II.Single/double digestion:③→⑦-BL21,②+⑩→BL21,④+⑥→BL21 [Qiyu
Tan][Sirui Dong][Rui Yao][Yuxiao Qin]

According to the results of agarose gel eletrophoresis,digest the
above 4 groups of plasmids except ④→NA overnight at room temperature

The system of digestion was as follows(units:μL)

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image340.png)

Digest overnigt at room temperature

III.IPTG induction:③→⑦-BL21,①→⑨-BL21 [Huixin Liang][Yuetong Ge]

We shake the bacteria solution obtained from ①→⑨-BL21 and ③→⑦-BL21
and induced them by IPTG for 4h, store other cloudy bacteria
solution

Discription Picture

Shake the bacteria before adding IPTG

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image341.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image342.png)

IV. PIck single colony and shake : ③→⑦-BL21, ①→⑪-BL21, ③→⑤-BL21,
①→⑨-BL21 [Huixin Liang][Yuetong Ge][Qiyu Tan]

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image343.png)

V. Store the bacteria : ③→⑦-BL21(7.20), ①→⑨-BL21(7.23),
④→⑧-BL21(7.23), ③→⑤-BL21(7.23), ③→⑦-BL21(7.25), ①→⑪-BL21(7.25),
③→⑤-BL21(7.25), ①→⑨-BL21(7.25)[Huixin Liang][Yuetong Ge][Qiyu Tan]

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image344.png)

Figure

Discription Picture

Mark the tube before store the bacteria

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image345.png)

VI. Transformation:②→⑫-BL21/BL21[Huixin Liang][Yuetong Ge][Qiyu Tan]

Because all the transformation about plasmid ② failed,so we
re-transformated and coated the plates

Follow protocol:Transformation

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image346.png)

Results of transformation (7.26 observed)

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image347.png)

2024.07.26 Friday

Outline:

I.Agarose gel electrophoresis:③→⑦-BL21,②+⑩→BL21,④+⑥→BL21[Qiyu
Tan][Sirui Dong]

II.Ultrisonic bacteria disruption, Fluorescence detection and
primary part of WB:③→⑦-BL21,①→⑨-BL21[All paricipation]

III.Shake bacteria::③→⑦-BL21/BL21,②→⑫-BL21[Huixin Liang][Yuetong Ge]

IV.Preparation of preparing competent cells:⑥-BL21,⑩-BL21[Huixin
Liang][Yueting Guo]

V.Preparation and verification of antibiotic[Huixin Liang]

①H01 with lac operator-pET-15b(pET-15b-lacO-H01-linker-EGFP)

②H05 with lac operator-pET-15b(pET-15b-lacO-H05-linker-EGFP)

③H01 without lac operator-pET-15b(pET-15b-H01-linker-EGFP)

④H05 without lac operator-pET-15b(pET-15b-H05-linker-EGFP)

⑤Input 01 without lac operator-pCOLADuet-1(pCOLADuet-input01)

⑥Input 05 without lac operator-pCOLADuet-1(pCOLADuet-input05)

⑦Input 01 without lac operator-pCDFDuet-1(pCDFDuet-input01)

⑧Input 05 without lac operator-pCDFDuet-1(pCDFDuet-input05)

⑨Input 01 with lac operator-pCOLADuet-1(pCOLADuet-lacO-input01)

⑩Input 05 with lac operator-pCOLADuet-1(pCOLADuet-lacO-input05)

⑪Input 01 with lac operator-pCDFDuet-1(pCDFDuet-lacO-input01)

⑫Input 05 with lac operator-pCDFDuet-1(pCDFDuet-lacO-input05)

Detailed Experiments:

I. Agarose gel electrophoresis:③→⑦-BL21,②+⑩→BL21,④+⑥→BL21[Qiyu
Tan][Sirui Dong]

Follow protocol:Agarose gel electrophoresis

The loading order and system are as follows(unit:μL)

Load 6 μL marker

Add 8μL 6x loading buffer to each sample,load the total 48μL

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image348.png)

II.Ultrisonic bacteria disruption, Fluorescence detection and
primary part of WB:③→⑦-BL21,①→⑨-BL21[All paricipation]

Follow protocol:Multi-Mode Microplate Reader

Ultrisonic bacteria disruption

1. Precipitate the suspended bacteria with 10ml PBS

2. Put the probe of the ultrasonic crusher into the heavy suspension
and give it an ice bath(The probe should not touch the wall or the
bottom, and should be cleaned with RO water in time after each
use;Change the ice in the ice bath in time)

3. After clicking "Work", each tube of bacteria is carried out 3
times, and each project is carried out 10 times (each work 10s, rest
40s).

4. Centrifuge the broken bacteria for 10min at 5000rpm

5.Each tube of supernatant is transferred to 4-5mL centrifugal tubes
(roughly 3ml in each centrifugal tube, not enough to fill 2.5ml)

6.Store the sorted supernatant in the refrigerator at -80℃ (bag
marked with supernatant protein, where the label + of the tube
should be →)

7.Start the machine, turn on the computer, open the software
spectrophotometer, select A280

8. Clean the upper and lower measurement parts with 2.5μL ddH2O, and
blot them dry with filter paper

9.Add 2μL ddH2O to the measuring part below the machine, cover the
lid, and the software is blank. Move the mouse away and the software
is measure to display the result. If the concentration is too high,
repeat step 3 until the result is displayed as 0.0x

10. Dry the filter paper, add 2μL supernatant to the measuring part
under the machine, and test each tube twice

11.Blot the filter paper, repeat step 2, clean the machine, shut
down the computer and software

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image349.png)

Fluorescence detection

1. Add the ③→⑦ IPTG+ 12μL supernatant to 138μLPBS, and add the
remaining 7.5μL supernatant to 142.5μL PBS, and mix well. The final
concentration is 1.8mg/ml

2. Add the protein sample to the 96-well plate (black) and measure
the fluorescence (488nm,525nm)

Resullts

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image350.png)

Primary part of WB

1.Sampling:

system as above empty for 10μL 1xbuffer (to prevent collapsing when
running glue) marker for 5μL buffer + 5μL marker original tube
concentration of 18ug/μL diluted five times, on the sample 10μL

90V constant voltage running glue, to be sampled on the strip
connected to a line and adjusted to 135V.

2.Transfer the film

use methanol for 3-5min activation soak the film

remove the glue on the line and the comb shape of the glue, with the
transfer of the mould liquid in the process has been often wet glue,
keep the glue wet

in accordance with the white board, sponge, two pieces of thick
filter paper, a piece of thin filter paper, the film, the glue, a
piece of thin filter paper, two thick filter paper, sponge, the
blackboard synthetic box

ice

to 200mA current to turn the film 2h. 200mA current to turn the
membrane for 2h

III.Attach milk, closed The membrane with bands will be placed in
milk, shaking for 30min

IV.Add primary antibody, overnight

Figure

DIscription Picture

Centrifuge the bacteria solution and get the precipitation before
ultrisonic disruption

Centrifuge the bacteria solution after ultrisonic disruption and get
the supernatant fluid

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image352.png)

III.Cultivation::③→⑦-BL21/BL21,②→⑫-BL21[Huixin Liang][Yuetong Ge]

Because we previously forgot to perform negative controls (without
IPTG and without input plasmid), so we use the rest bacteria
solution of ③→⑦-BL21 to preform IPTG induction again.

Cultured ③→⑦-BL21 and ② (two germplasm) →BL21, ③→BL21

All but ③→BL21 failed, possibly because the LB medium had been
reformulated

Figure

Discription Picture

The bacteria seem to be die after shaking

IV.Preparation of preparing competent cells:⑥-BL21,⑩-BL21[Huixin
Liang][Yueting Guo]

We precooled the materials we needed and prepare a mixture of
glycerol and CaCl2

V.Preparation and verification of antibiotic[Huixin Liang]

We prepared and verified a new 100mg/ml spectacularmycin

2024.07.27 Saturday

Outline:

I.The posterior part of WB:①→⑨-BL21+/-,③→⑦-BL21+[Yueting Guo]

II.Shaking/IPTG induction and store the bacteria :②+⑩→BL21 ④+⑥→BL21,
②→BL21, ③→⑦-BL21,③→BL21,⑥-BL21,⑩-BL21[Huixin Liang][Yuetong Ge][Qiyu
Tan][Sirui Dong][Rui Yao][Yuxiao Qin]

III.Preparation of competent cells:⑥-BL21,⑩-BL21[Huixin
Liang][Yuetong Ge]

IV.Agarose gel nucleic acid eletrophoresis:verification of
EcoRI[Qiyu Tan][Sirui Dong][Rui Yao][Yuxiao Qin]

①H01 with lac operator-pET-15b(pET-15b-lacO-H01-linker-EGFP)

②H05 with lac operator-pET-15b(pET-15b-lacO-H05-linker-EGFP)

③H01 without lac operator-pET-15b(pET-15b-H01-linker-EGFP)

④H05 without lac operator-pET-15b(pET-15b-H05-linker-EGFP)

⑤Input 01 without lac operator-pCOLADuet-1(pCOLADuet-input01)

⑥Input 05 without lac operator-pCOLADuet-1(pCOLADuet-input05)

⑦Input 01 without lac operator-pCDFDuet-1(pCDFDuet-input01)

⑧Input 05 without lac operator-pCDFDuet-1(pCDFDuet-input05)

⑨Input 01 with lac operator-pCOLADuet-1(pCOLADuet-lacO-input01)

⑩Input 05 with lac operator-pCOLADuet-1(pCOLADuet-lacO-input05)

⑪Input 01 with lac operator-pCDFDuet-1(pCDFDuet-lacO-input01)

⑫Input 05 with lac operator-pCDFDuet-1(pCDFDuet-lacO-input05)

Detailed Experiments:

I.The posterior part of WB:①→⑨-BL21+/-,③→⑦-BL21+[Yueting Guo]

We continue to finish the rest steps(after recover the primary
antibody overnight) of WB

1.Wash the membrane 3 times with TBST.

Add a small amount of TBST to the cassette without overflowing the
membrane, shake on a shaker (110-120) for 10 min, discard the TBST
in the cassette and add new TBST.

Repeat three times without adding TBST for the third time.

Thaw the secondary antibody in water in advance, add the secondary
antibody and shake on a shaker (50-60) for one hour to recover the
secondary antibody.

Wash the membrane three times with TBST again.

2. Developing

Put the film into the black box of exposure solution, cover the box
with a lid and shake it slightly for a few times to develop the
exposure.

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image354.png)

II.Shake,IPTG induce or not and store the bacteria :②+⑩→BL21
④+⑥→BL21, ②→BL21, ③→⑦-BL21,③→BL21,⑥-BL21,⑩-BL21[Huixin
Liang][Yuetong Ge][Qiyu Tan][Sirui Dong][Rui Yao][Yuxiao Qin]

Shake bacteria

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image355.png)

Discard the ungrown bacterial solution:2 of ②+⑩→BL21,1 of ④+⑥→BL21,1
of ②→BL21,2 of ③→BL21

For the bacteria shaken for IPTG induction,add 40μL IPTG(c=0.5M) to
each conical flask and shake for another 4h

Centrifuge the bacteria solution after shaking and store at -80℃
waiting for ultrasonic crushing

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image356.png)

Store the bacteria

Shake the bacteria to prepare for the IPTG induction tomorrow

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image357.png)

Store the bacteria

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image358.png)

Figure

Discription Picture

Store the bacteria as the picture

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image359.png)

Centrifuge the bacteria solution after IPTG induction/shaking

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image360.png)

Pick single colony and shake

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image361.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image362.png)

III.Preparation of competent cells:⑥-BL21,⑩-BL21[Huixin
Liang][Yuetong Ge]

Follow protocol:Preparation of competent cells

IV.Agarose gel nucleic acid eletrophoresis:verification of
EcoRI[Qiyu Tan][Sirui Dong][Rui Yao][Yuxiao Qin]

To verify which EcoRI can lead to wrong results,we use 3 enzyme to
run agarose gel

Follow protocol:Agarose gel electrophoresis

Use 1% agarose gel

Run at 135V for 40min

The loading order and system were as folows(unit:μL)

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image363.png)

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image364.png)

Analysis

EcoRI-S had some problem which can lead to wrong results

2024.07.28 Sunday

Outline:

I.Ultrisonic bacteria disruption,fluorescence detection and the
primary part of WB:④+⑥→BL21,③→⑦-BL21 , ②-BL21[Saiyu Luo][Qiyu
Tan][Sirui Dong][Rui Yao][Yuxiao Qin]

II.Store the
bacteria:①→BL21、②(extracted)→BL21,③→BL21,②(extracted)→⑫-BL21
[Huixin Liang][Yuetong Ge][Yueting Guo]

III.IPTG
induction:①→BL21、②(extracted)→BL21,③→BL21,②(extracted)→⑫-BL21
[Huixin Liang][Yuetong Ge][Yueting Guo]

IV.Cultivation:④→BL21,④→⑧-BL21 [Huixin Liang][Yuetong Ge][Yueting
Guo]

V.Preparation of LB medium[Qiyu Tan][Sirui Dong]

①H01 with lac operator-pET-15b(pET-15b-lacO-H01-linker-EGFP)

②H05 with lac operator-pET-15b(pET-15b-lacO-H05-linker-EGFP)

③H01 without lac operator-pET-15b(pET-15b-H01-linker-EGFP)

④H05 without lac operator-pET-15b(pET-15b-H05-linker-EGFP)

⑥Input 05 without lac operator-pCOLADuet-1(pCOLADuet-input05)

⑦Input 01 without lac operator-pCDFDuet-1(pCDFDuet-input01)

⑧Input 05 without lac operator-pCDFDuet-1(pCDFDuet-input05)

⑫Input 05 with lac operator-pCDFDuet-1(pCDFDuet-lacO-input05)

Detailed Experiments:

I.Ultrisonic bacteria disruption,fluorescence detection and the
primary part of WB:④+⑥→BL21,③→⑦-BL21 , ②-BL21[Saiyu Luo][Qiyu
Tan][Sirui Dong][Rui Yao][Yuxiao Qin]

Follow protocol:Ultrisonic bacteria disruption ,Fluoredcence
detection ,WB

Results of fluoredcence detection

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image365.png)

Figure

Discription Picture

Add 10ml PBS and mix well

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image366.png)

II. Store the
bacteria:①→BL21、②(extracted)→BL21,③→BL21,②(extracted)→⑫-BL21
[Huixin Liang][Yuetong Ge][Yueting Guo]

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image367.png)

Figure

Discription Picture

Store the bacteria

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image368.png)

III.IPTG
induction:①→BL21、②(extracted)→BL21,③→BL21,②(extracted)→⑫-BL21
[Huixin Liang][Yuetong Ge][Yueting Guo]

Shake at 220 rpm for 4h before add IPTG

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image369.png)

Results

①→BL21 failed to grow,others grew successfully

Add 40μL IPTG(c=0.5M) to one conical flask of each group,and shake
for another 4h at 220rpm,37℃

Centrifuge the bacteria solution at 5000rpm,10min at a time,store
the precipitation at -80℃

III. Cultivation:④→BL21,④→⑧-BL21 [Huixin Liang][Yuetong Ge][Yueting
Guo]

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image370.png)

V.Preparation of LB medium[Qiyu Tan][Sirui Dong]

Follow protocol:Preparation of LB Medium

We prepare 2.8L LB in total

2024.07.29 Monday

Outline:

I.Ultrisonic bacteria disruption,:③-BL21 IPTG+/-,④→⑧-BL21
IPTG-,④→BL21(IPTG-)[Qiyu Tan][Sirui Dong][Rui Yao]

II.Fluorescence detection:③→⑦-BL21 IPTG+/-,③-BL21 IPTG+/-[Qiyu
Tan][Sirui Dong][Rui Yao]

III.Primary part of WB:③→⑦-BL21 IPTG+/-,③-BL21 IPTG+/-[Sirui Dong]

IV.Store the bacteria and IPTG induction:④→⑧-BL21 ,④→BL21[Huixin
Liang][Yuetong Ge]

③H01 without lac operator-pET-15b(pET-15b-H01-linker-EGFP)

④H05 without lac operator-pET-15b(pET-15b-H05-linker-EGFP)

⑧Input 05 without lac operator-pCDFDuet-1(pCDFDuet-input05)

Detailed Experiments:

I.Ultrisonic bacteria disruption:③→BL21 IPTG+/-,④→⑧-BL21
IPTG-,④→BL21(IPTG-)[Qiyu Tan][Sirui Dong][Rui Yao]

Follow protocol:Ultrisonic bacteria disruption

IV. Fluorescence detection:③→⑦-BL21 IPTG+/-,③→BL21 IPTG+/-[Qiyu
Tan][Sirui Dong][Rui Yao]

Follow protocol:Fluorescence detection

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image371.png)

Results

III.Primary part of WB:③→⑦-BL21 IPTG+/-,③-BL21 IPTG+/-[Sirui Dong]
[Saiyu Luo][Yuxiao Qin]

Follow protocol: WB,Preparation of running buffer and trans buffer

Figure

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image372.png)

Discription Picture

Prepare the 10x running buffer and 10x trans buffer

IV.Store the bacteria and IPTG induction:④→⑧-BL21 ,④→BL21[Huixin
Liang][Yuetong Ge]

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image373.png)

Figure

Discription Picture

process

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image374.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image375.png)

2024.07.30 Tuesday

Outline:

I.Ultrisonic bacteria disruption:④→⑧-BL21 IPTG+/-;④→BL21 IPTG+/-
[all participants]

II.Primary part of WB and coomassie brilliant blue:④→⑧-BL21
IPTG+/-;④→BL21 IPTG+/-,③→⑦-BL21 IPTG+/-;③→BL21 IPTG+/-[all
participants]

III.Maxiprep for plasmids and digestion:④→⑧-BL21[Rui Yao][Yuxiao
Qin]

IV.Shake the bacteria:④→⑧-BL21;④→BL21;①→BL21 [Huixin Liang][Yuetong
Ge]

①H01 with lac operator-pET-15b(pET-15b-lacO-H01-linker-EGFP)

③H01 without lac operator-pET-15b(pET-15b-H01-linker-EGFP)

④H05 without lac operator-pET-15b(pET-15b-H05-linker-EGFP)

⑦Input 01 without lac operator-pCDFDuet-1(pCDFDuet-input01)

⑧Input 05 without lac operator-pCDFDuet-1(pCDFDuet-input05)

Detailed Experiments:

I.Ultrisonic bacteria disruption and fluorescence detection:④→⑧-BL21
IPTG+/-;④→BL21 IPTG+/-[all participants]

Follow protocol:Ultrisonic bacteria disruption and fluorescence
detection

Results of ultrisonic bacteria disruption

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image376.png)

Results of fluorescence detection

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image377.png)

II.Primary part of WB and coomassie brilliant blue:④→⑧-BL21
IPTG+/-;④→BL21 IPTG+/-,③→⑦-BL21 IPTG+/-;③→BL21 IPTG+/-[all
participants]

Follow protocol:Western Blot and Coomassie staining of the gel

Prepare sample of SDS-PAGE as follows

1.Prepare 5x sample system

Target: c=18ug/μL 100μL

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image378.png)

2.Prepare 1X sample

Target: 3.6ug/μL 100μL

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image379.png)

3. prepare 1X sample directly

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image380.png)

4. After preparation, heat at 100℃ for 15 mins, then cool down.

Total 2 groups, each group run two pieces of the same glue, a total
of 4 pieces of glue, sample order is as follows (from left to right)

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image381.png)

Preparation of Coomassie bright blue dye and eluent

Follow protocol:Coomassie brilliant blue dye and destaining solution

We prepare 200ml 0.25% coomassie brilliant blue dye and 1L eluent in
total

III.Maxiprep for plasmids and digestion:④→⑧-BL21[Rui Yao][Yuxiao
Qin]

Follow protocol:Vigorous plasmid maxiprep and restriction digest of
plasmid DNA

Results of maxiprep

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image382.png)

Digest system

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image383.png)

Digest overnight at room temperature

IV. Shake the bacteria:④→⑧-BL21;④→BL21;①→BL21 [Huixin Liang][Yuetong
Ge]

We shake bacteria in 15ml tube for IPTG induaction tomorrow

Shake at 220rpm,37℃

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image384.png)

2024.07.31 Wednesday

Outline:

I.Subsequent part of WB:④→⑧-BL21 IPTG+/-;④→BL21 IPTG+/-,③→⑦-BL21
IPTG+/-;③→BL21 IPTG+/-[Huixin Liang][Yuetong Ge][Yueting Guo]

II.Coomassie brilliant blue color destaining:④→⑧-BL21 IPTG+/-;④→BL21
IPTG+/-,③→⑦-BL21 IPTG+/-;③→BL21 IPTG+/-[Qiyu Tan][Sirui Dong]

III.IPTG induction:④→⑧-BL21,④→BL21,①→BL21[Huixin Liang][Yuetong
Ge][Yueting Guo]

IV.Ultrisonic bacteria disruption:①→BL21,②→⑫-BL21 [Huixin
Liang][Yuetong Ge] [Yueting Guo][Qiyu Tan][Sirui Dong]

V.Fluorescence detection:②→⑫-BL21[Qiyu Tan][Sirui Dong]

VI.Agarose gel electrophoresis and digestion[Rui Yao][Yuxiao Qin]

VII.Cultivation:⑧-BL21 [Rui Yao][Yuxiao Qin]

①H01 with lac operator-pET-15b(pET-15b-lacO-H01-linker-EGFP)

②H05 with lac operator-pET-15b(pET-15b-lacO-H05-linker-EGFP)

③H01 without lac operator-pET-15b(pET-15b-H01-linker-EGFP)

④H05 without lac operator-pET-15b(pET-15b-H05-linker-EGFP)

⑦Input 01 without lac operator-pCDFDuet-1(pCDFDuet-input01)

⑧Input 05 without lac operator-pCDFDuet-1(pCDFDuet-input05)

⑫Input 05 with lac operator-pCDFDuet-1(pCDFDuet-lacO-input05)

Detailed Experiments:

I.Subsequent part of WB:④→⑧-BL21 IPTG+/-;④→BL21 IPTG+/-,③→⑦-BL21
IPTG+/-;③→BL21 IPTG+/-[Huixin Liang][Yuetong Ge][Yueting Guo]

Follow protocol:Western Blot

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image385png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image386.png)

II.Coomassie brilliant blue color destaining:④→⑧-BL21 IPTG+/-;④→BL21
IPTG+/-,③→⑦-BL21 IPTG+/-;③→BL21 IPTG+/-[Qiyu Tan][Sirui Dong]

Follow protocol: Coomassie staining of the gel

V. Store bacteria and IPTG induction:④→⑧-BL21,④→BL21,①→BL21[Huixin
Liang][Yuetong Ge][Yueting Guo]

Store bacteria before induction

Add 1ml bacteria solution from 15ml tube and shake at 220rpm for 4h
before induction

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image387.png)

Because of non-growth,group ④→BL21 shake for another 1h20min before
adding IPTG

Add 40μL IPTG(c=0.5M) to each conical flask and shake for another 4h

Centrifuge the bacteria solution at 5000rpm,10min at a time,store
the precipitation at -80℃

IV.Ultrisonic bacteria disruption:①→BL21,②→⑫-BL21[Qiyu Tan][Sirui
Dong][Huixin Liang][Yuetong Ge][Yueting Guo]

Follow protocol:Ultrasonic disruption of bacteria

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image388.png)

V.Fluorescence detection:③→⑦-BL21/BL21,④→⑧-BL21/BL21,②→⑫-BL21/BL21
[Qiyu Tan][Sirui Dong]

dilute sample to 1.8ug/μL total volume=650μL

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image389.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image390.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image391.png)

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image392.png)

VI.Agarose gel electrophoresis and digestion:group③→⑦-BL21 and group
④→⑧-BL21 [Rui Yao][Yuxiao Qin]

Follow protocol:Agarose gel electrophoresis and digestion

Loading order and system(unit:μL)

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image393.png)

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image394.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image395.png)

Due to the unsited bands, digest again to prepare 8.1
electrophoresis

System of digestion

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image396.png)

Digest overnight at room temperature

VII.Cultivation:⑧-BL21 [Rui Yao][Yuxiao Qin]

Because of the few remaining plasmids, shake ⑧ bacteria for the Maxi
plasmid extraction next day

Add 500μL bacteria solution to 200ml LB

The concentration of stc is 100ug/ml

Shake at 220 rpm overnight

2024.08.01 Thursday

Outline:

I. Ultrasonic bacteria disruption:④→⑧-BL21 IPTG+/-;④→BL21 IPTG+/-

[Huixin Liang][Yuetong Ge] [Qiyu Tan][Sirui Dong]

II. Fluorescence detection:④→⑧-BL21 IPTG+/-;④→BL21 IPTG+/-,①→⑨-BL21
IPTG+/-,①→BL21 IPTG+/- [Qiyu Tan][Sirui Dong]

III. WB(Part1):④→⑧-BL21 IPTG+/-;④→BL21 IPTG+/-,③→⑦-BL21
IPTG+/-;③→BL21 IPTG+/- [Huixin Liang][Yuetong Ge]

IV. Coomassie brilliant blue :④→⑧-BL21 IPTG+/-;④→BL21
IPTG+/-,③→⑦-BL21 IPTG+/-;③→BL21 IPTG+/- [Huixin Liang][Yuetong Ge]

V. Agarose gel electrophoresis:③,⑦,③→⑦-BL21,④,⑧,④→⑧-BL21 [Rui
Yao][Yuxiao Qin]

VI. Digestion:③,⑦,③+⑦ [Rui Yao][Yuxiao Qin]

VII. Maxiprep plasmid :⑧ [Rui Yao][Yuxiao Qin]

I. Ultrasonic bacteria disruption, Fluorescence detection and
primary part of SDS-PAGE:④→⑧-BL21 IPTG+/-;④→BL21 IPTG+/-,①→⑨-BL21
IPTG+/-,①→BL21 IPTG+/-

Follow protocol:Ultrasonic disruption of bacteria,Multi-Mode
Microplate Reader ,WB

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image397.png)

(1)Protein concentration:

(2) Fluorescence detection

Dilute protein sample to 1.8μg/μL

then Load each protein sample in four wells, 400μL per well,
distributed in A1~H4

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image398.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image399.png)

(3) WB sample making:

1X sample : 3.6μg/μL, 100μL System

Heat the sample at 100℃ for 5min and store at -20℃ for WB tomorrow

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image400.png)

Loading order as follows:

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image401.png)

(4) SDS-PAGE

Loading order as follows:

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image402.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image403.png)

II. Agarose gel electrophoresis:③,⑦,③→⑦-BL21,④,⑧,④→⑧-BL21

Follow protocol: Agarose gel electrophoresis

Use 1% agarose gel

Run at 135V for 40min

The loading order and system were as folows(unit:μL)

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image404.png)

Result

If the band is correct, the transformation of ③+⑦ and ④+⑧ is
successful. And the band is more distinct.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image405.png)

III. Digestion:③,⑦,③+⑦

The enzyme digestion system is as follows

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image406.png)

Digest at 37℃ for 30min

IV. Maxiprep plasmid :⑧

Follow protocol:Vigorous Plasmid Maxiprep

The concentration:

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image407.png)

Handwriting:

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image408.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image409.png)

2024.08.02 Friday

Outline:

I .The subsequent part of WB:④⑧series (7.31 induction) [Huixin
Liang][Yuetong Ge][Yueting Guo]

II. The subsequent part of SDS-PAGE :③⑦series [Qiyu Tan][Sirui Dong]

III. Agarose gel electrophoresis :③⑦series [Rui Yao][Yuxiao Qin]

Detailed Experimental Record

I. Preparation of blocking buffer(5% skim milk powder) and secondary
antibody

Follow protocol: Western blot

1. 5% milk(blocking buffer) preparation

Weigh 2.5g skim milk powder and add it into a 50mL centrifuge tube.
Set the volume to 50mL by TBST.Mix upside down, further mix on the
shaker(100rpm)

2. 1% blocking buffer

Take 2mL 5% skim milk powder in 15mL centrifuge tube and use TBST to
adjust volume to 10mL

3. Preparation of secondary antibodies

Add 5μL Goat Anti-Rabbit IgG H&L/HRP into 1% blocking buffer(
1:2000).

II. The subsequent part of WB

Follow protocol: Western Blot

Analysis:

Basically consistent with the results of fluorescence assay, the
expression of IPTG+ group was significantly higher than that of
IPTG- group, ④→BL21 IPTG+ was abnormally higher

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image410.png)

III Complete the SDS-PAGE Coomasil bright blue elusion

Follow protocol: Coomassie staining of the gel

Result:

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image411.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image412.png)

1. Bands close to the size of GFP protein appeared in the correct
position in both groups ③→⑦ and ④→⑧, and the GFP carried by the
plasmid was successfully expressed.

2. In groups ③→⑦, the expression of GFP protein in H01without lac
was significantly different under IPTG induction and with or without
input sequence, but the band signal of H01 IPTG- was higher and
slightly less than 27kDa, which was inconsistent with the results of
the microplate reader. The reason is unclear.

3. In groups ④→⑧, the effect of IPTG induction was obvious, but the
effect of input on the GFP expression of H05 sequence was not high,
which was consistent with the results of the Multi-mode Microplate
Reader.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image413.png)

IV Perform ③-⑦ series agarose gel electrophoresis

Follow protocol: Agarose gel electrophoresis

120mL 1XTAE should be heated to 100mL to make the glue to reach the
appropriate concentration, otherwise the glue concentration is low,
the band may be a little loose

It is placed on the -20℃ refrigerator in room 1008

Electrophoresis at 135v,45min

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image414.png)

Handwriting

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image415.png)

2024.08.05 Monday

Outline:

I. Cultivation:①→⑨-BL21 and ②(Maxi prep)→ ⑫-BL21 [Huixin
Liang][Yuetong Ge]

II. Digestion:④-⑥;②-⑩;⑧ [Rui Yao][Yuxiao Qin]

Detailed Experimental Record

I. Cultivation:①→⑨-BL21 and ②(Maxi prep)→ ⑫-BL21

Take two bottles of 200mL LB medium and add 200μL Amp+200μL
Stc/200μL Amp+200μLkan (the concentration of antibiotics is
100mg/mL), shake well

Take two 15mL tubes and mark them as follows(Add the corresponding
5mL LB medium):

①→⑨-BL21 A+K+ Bacteria 1A (7.25) 8.5 Shake

② → ⑫-BL21 A+S+ Bacteria 1A (7.25) 8.5 Shake

Add 1mL of the corresponding LB-antibiotic (as shown in the figure)
and put it into the shaking incubator(220rpm, 37℃).

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image416.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image417.png)

After 1.5h, the bacterial solution was found to be turbid, we
absorbed 3mL bacterial solution from each tube and added them to the
above 200mL LB medium at 220rpm and shaked overnight at 37℃.

II. Digestion:④-⑥;②-⑩;⑧

Systems are shown below

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image418.png)

Put the system under the room temperature, digest overnight.

Handwriting

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image419.png)

2024.08.06 Tuesday

Outline:

I.Agarose gel electrophoresis ④-⑥;②-⑩;⑧ series [Rui Yao][Yueting
Guo]

II. Maxiprep plasmid: ①→⑨-BL21 and ②(Maxiprep)→ ⑫-BL21 [Rui
Yao][Yueting Guo]

III. Digestion:①→⑨; ②-⑩; ②+⑫ [Rui Yao][Yueting Guo]

Detailed Experimental Record

I. Agarose gel electrophoresis ④-⑥;②-⑩;⑧ series

Follow protocol: Agarose gel electrophoresis

Use 1% agarose gel

Run at 135V for 40min

The load order and system are as follows(unit:μL)

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image420.png)

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image421.png)

II. Maxiprep plasmid:①→⑨-BL21 and ②(Maxi prep)→ ⑫-BL21

Follow protocol:Vigorous plasmid maxiprep

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image422.png)

Note:

①→⑨ plasmid crude extract about 1mL, because the previous step is
not completely drained

②→⑫ bacteria was fully lysed but still transparent

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image423.png)

III. Digest:①→⑨;②-⑩;②+⑫

Follow protocol:Agarose gel electrophoresis

The system are as follows:

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image424.png)

Put the system under the room temperature, digest overnight.

Handwriting

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image425.png)

2024.08.07 Wednesday

Outline:

I.Agarose gel electrophoresis of ②from →⑫-BL21 series, ①→⑨-BL21
series, ②+⑩→BL21 series [Rui Yao][Yueting Guo][Yueting Guo]

II. Shake bacteria ①→⑨-BL21 [Huixin Liang][Yuetong Ge]

Detailed Experiments

I. Agarose gel electrophoresis of ② from → -BL21 series, ①→⑨-BL21
series, ②+⑩→BL21 series

Follow protocol:Agarose gel electrophoresis

Use 1% agarose gel

Run at 135V for 40min

The load order and system are as follows(unit:μL)

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image426.png)

Result:

The concentration of ①+⑨ is too low, resulting in no banding. So,we
shaked the bacteria again.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image427.png)

II. Shake bacteria ①→⑨-BL21

1. Take 1 bottles of 200mL LB medium and add 200μL Amp+200μL kan
(the concentration of antibiotics is 100mg/mL), shake 2h

2. Take 2 x15mL tubes and mark them as follows:

①→⑨-BL21 A+K+ Bacteria 1B (7.25) 8.7 Shake

①→⑨-BL21 A+K+ Bacteria 2A (7.25) 8.7 Shake

Add 5mL corresponding LB-medium

3. Add 500μL of the corresponding LB-antibiotic medium (as shown in
the figure) and put it into the shaking incubator(220rpm, 37℃).

4.After 2h, the bacterial solution was found to be turbid, and
absorb 4mL bacterial solution from ①→⑨-BL21 A+K+ Bacteria 2A (7.25)
8.7 ,added to the above 200mL LB medium at 220rpm and shake
overnight at 37℃

Handwriting

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image428.png)

2024.08.08 Thursday

Outline:

I. Maxiprep plasmid :①→⑨ [Rui Yao][Yuxiao Qin]

II. Preparation of LB medium [Rui Yao][Yuxiao Qin][Yueting Guo]

III. Digestion:①→⑨series [Rui Yao][Yuxiao Qin][Yueting Guo]

Detailed Experiments

I. Maxiprep plasmid :①→⑨

Follow protocol:Vigorous plasmid maxiprep

Results

The concentration of Group ①→⑨ is 60.8ng/μL

II. Digestion:①→⑨series

Systems are shown below

Digest under room temperature overnight.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image429.png)

Handwriting

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image430.png)

2024.08.09 Friday

Outline:

I. Agarose gel electrophoresis ①→⑨-BL21 series [Rui Yao][Yueting
Guo]

II. Paper part

Write notebook[Huixin Liang][Yuetong Ge]

Write part page [Qiyu Tan]

Write Result page[Sirui Dong]

Write protocol page [Yueting Guo]

Detailed Experiments

Follow protocol: Agarose gel electrophoresis

Use 1% agarose gel

Run at 135V for 40min

The load order and system are as follows(unit:μL)

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image431.png)

Result:

All the bands are expected.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image432.png)

Handwriting

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image433.png)

2024.08.12 Monday

Outline:

I. Digestion:①-⑫ all plasmids [Rui Yao][Yueting Guo]

I. Formulate complete medium [Sirui Dong][Saiyu Luo]

II. Cell recovery:786-O [Sirui Dong][Saiyu Luo][Huixin
Liang][Yuetong Ge]

Detailed Experiments

I. Digestion:①-⑫ all plasmids

Systems are shown below

Digest under room temperature overnight

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image434.png)

II. Complete medium:

The base medium for this cell line is RPMI-1640 Medium.

To make the complete growth medium, add the following components to
the base medium: fetal bovine serum to a final concentration of 10%.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image435.png)

III. Thawing of Frozen Cell Lines: 786-O

1. Collect cells:786-O from liquid nitrogen storage wearing
appropriate personal protective equipment and transfer to the
laboratory in a container of liquid nitrogen or on dry ice.

2. Quickly transfer the freezing tube to a 37°C water bath until
only one or two small ice crystals, if any, remain 2 minutes. It is
important to thaw rapidly to minimize any damage to the cell
membranes.

Note: Do not totally immerse the freezing tube as this may increase
the risk of contamination.

3. Pellet the cells by centrifugation at 150 x g for 5 minutes and
resuspend the cell pellet in fresh medium using the 12mL volume to
achieve the correct seeding density in 10cm petri dish.

Handwrting

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image436.png)

2024.08.13 Tuesday

Outline:

I. Digestion: ⑦⑧⑨⑫(again) [Rui Yao][Yueting Guo]

II. Agarose gel electrophoresis: ①-⑫ [Rui Yao][Yueting Guo]

III. Culture the cells 786-O in vitro [Huixin Liang][Yuetong Ge]

Detailed Experiments

I. Agarose gel electrophoresis: ①-⑫

Follow protocol: Agarose gel electrophoresis

Use 1% agarose gel

Run at 135V for 40min

The load order and system are as follows(unit:μL)

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image437.png)

Result:

The location of the bands is roughly correct, and some bands are not
aesthetically expected (⑨ do not run away, ⑦⑧⑩ are too bright).

The result is as follows:

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image438.png)

II. Digestion:⑦⑧⑨⑫ again according to the results

Systems are shown below

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image439.png)

Put the system under the room temperature, digest overnight.

IV. Culture the cells 786-O in vitro

Follow protocol: Cell lines

1. Observe the state of the cells, they grow well.

2. Discard the supernatant. Wash once with 5 mL PBS carefully.

3. Add the medium 12mL, place them in the incubator.

Handwriting

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image440.png)

Date:2024.08.14 Wednesday

Outline:

I. Agarose gel electrophoresis: ⑦⑧⑨⑫ [Rui Yao][Yueting Guo]

I.Passage the cells 786-O [Sirui Dong][Saiyu Luo] [Qiyu Tan][Huixin
Liang][Yuetong Ge]

Detailed Experiments

I. Agarose gel electrophoresis: ①-⑫

Follow protocol: Agarose gel electrophoresis

Use 1% agarose gel

Run at 135V for 40min

The load order and system are as follows(unit:μL)

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image441.png)

Result:

The location of the bands is roughly correct.

The picture is as follows:

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image442.png)

II. passage

Digest the cells:

① Use a pipette to aspirate out the waste liquid in the culture dish
under negative pressure. Add 3mL PBS to rinse and then suck out the
liquid again

② Add trypsin digestion solution 2mL.Incubate at 37°C for 2 minutes.

③ Add 2mL DMEM, rinse the bottom of the culture dish and collect
cells.

④Centrifuge cells at 1000rpm for 5min.

⑤ Discard the supernatant, add PBS again, centrifuge again and
remove the supernatant

⑥ Add 1mL DMEM and mix well.

⑦ Dilute the cells to a general cell density of 5 X 103 cells per
well

Handwriting

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image443.png)

2024.08.16 Friday

Outline:

I. Transwell and wound healing (Day1)

[Sirui Dong][Saiyu Luo] [Qiyu Tan][Huixin Liang][Yuetong Ge]

II.Paper Part

I Write notebook page [Huixin Liang][Yuetong Ge][Yueting Guo]

II Write Parts page [Qiyu Tan]

III Design the wet lab parts of wiki [Sirui Dong]

IV Write Protocol page [Rui Yao][Yuxiao Qin], [Yueting Guo] helped

V Design experiments [Yuetong Ge]

VI Connect with model group [Qiyu Tan][Rui Yao][Yuetong Ge]

Detailed Experiments

I.Prepare the overexpression conditions:

Groups:mimic-142-3p ; inhibitor-142-3p ;mimic-210-3p ;
inhibitor-210-3p

1. Add OPti-Medium and RNA iMax into three EP tubes , (100μl
OPti-Medium and 1μl RNA iMax each ),mix well,and hold at room
temparature for 5min

2. Dilute
mimic-142-3p/inhibitor-142-3p/mimic-210-3p/inhibitor-210-3p with
DEPC H2O

(1OD + 125 μL DEPC H2O);

Dilute control with DEPC H2O (0.5OD Control + 125 μl DEPC H2O)

3. Separately add 1μL mimic-142-3p, 1μL inhibitor-142-3p,1μL
mimic-210-3p ;1μL inhibitor-210-3p,1μL control into aforesaid six EP
tubes, mix well, and hold at room temparature for 20min.

II. Transwell

Follow protocol:Transwell

1. Prepare - Matrigel: empty medium DMEM = 1:4

Add 40 μL of prepared Matrigel to the Transwell

2.Leave it at 37°C until Matrigel solidifies.About 45min.

3.Take out the cultured cells, discard the medium, digest the cells
with trypsin. After termination of the medium containing serum,
collect the cells in a 15ml centrifuge cylinder and centrifuge it at
1000rpm for 5min.

4.Discard the supernatant, gently resuspend the cells with 5 ml of
PBS for washing, and centrifuge it at 1000 rpm for 5min.

5.Resuspend the cells with 2 ml of serum-free medium for cell
counting. The number of cells is 5×104. The volume of cells and
serum-free medium is 100 μL in total.

6. In the empty 24-well plate, add 650 μl of culture medium with 20%
serum at the bottom of each well. Sit the chamber on the top and add
100 μl of mixed serum-free cell mixture into the upper chamber.

7. Add the cells vertically. Be careful not to touch the upper
surface of the chamber.

8. Add reagents to upper chamber and lower chamber :

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image444.png)

(1)After the upper chamber glue has set,add 100μL cell suspension to
per well of it,add 100μL overexpression conditions to per well of
it.

(2)Add 650μL DMEM containing 20% serum to each well of the lower
chamber(500μLDMEM+150μL serum)

9.Culture it at 37℃ for 48h.

III. Wound Healing

Follow protocol:Wound Healing

1.Take a six-well plate and mark three parallel lines on the bottom.
Label the corners with information about each well.

2.Cells were plated in a general cell density of 5X105 cells per
well.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image445.png)

Date:2024.08.17 Saturday

Outline:

I. Transwell and wound healing (Day2)

[Sirui Dong][Saiyu Luo] [Qiyu Tan][Huixin Liang][Yuetong Ge]

Detailed Experiments

Wound healing

Follow protocol: Wound Healing

1.The cells are full grown, the six-well plate is withdrawn and
three parallel lines are drawn with the tips along the previously
drawn straight line.

2.Wash slowly with PBS and add 3mL of serum-free medium. Select the
spot for photographing with an inverted microscope, and mark it as
0h.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image447.png)

Figure1:Wound Healing result of exosomes containing miR-142-3p in
786-o cells.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image448.png)

Figure2:Wound Healing result of exosomes containing miR-210-3p in
786-o cells.

2024.08.18 Sunday

Outline:

I. Transwell and Wound healing (Day3)

[Sirui Dong][Saiyu Luo] [Qiyu Tan][Huixin Liang][Yuetong Ge]

II.Paper work(This week)

Write notebook[Huixin Liang][Yuetong Ge]

Write part page [Qiyu Tan]

Write Result page[Sirui Dong]

Write protocol page [Yueting Guo]e[Rui Yao][Yuxiao Qin],

Detailed Experiments

I .Transwell

Follow protocol:Transwell

1. Fix cells:

Add 850 μL 4% paraformaldehyde to the empty wells. Place the
chambers into

the wells and fix for 20min.

2. Giemsa staining:

Fix the cells in methanol for 10 min, discard the fixative and rinse
the cells with distilled water.

The Giemsa stain was diluted with PBS (Giemsa stain:PBS=1:9). Add
850μL of Giemsa stain to each well ,and stain with wet box at 37℃
for 1h or overnight at room temperature.

3. Rinse well with distilled water and place under the microscope.

4. Invasive cell counting: Randomly select ③-⑤ fields of view under
the microscope and count the number of cells migrating to the lower
side of the membrane.

Result:

Transwell result of miR-142-3p in 786-O cells. 786-O (PUMC) is human
kidney homo cells, which simulate the cancer environment to test
function of miR-142-3p invasion.

Transwell result of miR-210-3p in 786-O cells. 786-O (PUMC) is human
kidney homo cells, which simulate the cancer environment to test
function of miR-210-3p invasion.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image450.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image451.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image453.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image452.png)

II Wound healing

Follow protocol:Wound healing

1.Wash slowly with PBS and add 3mL of serum-free medium. Select the
spot for photographing with an inverted microscope, and mark it as
24h.

2024.08.19 Monday

Outline:

I. Wound Healing (Day3)

[Sirui Dong][Saiyu Luo] [Qiyu Tan][Huixin Liang][Yuetong Ge]

Detailed Experiments

II Wound healing

Follow protocol:Wound healing

1.Wash slowly with PBS and add 3mL of serum-free medium. Select the
spot for photographing with an inverted microscope, and mark it as
48h.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image448.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image454.png)

2024.08.22 Thursday

Outline:

I. Preparation of LB medium [Qiyu Tan]

II.Paper work

Detailed Experiments

I. Preparation of LB medium

Follow the protocol:LB liquid medium

(Total volume = 1.3 L, 5 flasks for liquid medium, 3 flasks for
solid medium)

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image456.png)

1. Add the specified amounts of tryptone, NaCl, and yeast extract to
a graduated container. Then, add RO H2O until the total volume
reaches 1.3 liter.

2. Stir them at 440r/min on a magnetic stirrer, until the solution
are completely dissolved.

3. Pour 200mL liquid into each 500mL conical flasks, pour 100mL
liquid into each 250mL conical flasks. There are 5 large
flasks(500mL), 3 small flasks(250mL).

4. Add 1.5g agarose powder to each small flasks. (Agar: Baygene,
LOT: 222518)

5. Sterilize the medium by autoclave sterilizer for 20 min at 121℃.

IV. Prepare LB solid medium

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image457.png)

1. Take antibiotics from -20℃ fridge.

2. Each antibiotic was added in 50μL to the corresponding conical
flask, concentration of antibiotics is all 100 mg/mL.

3. Pour LB liquid medium to the plates, then cool them at room
temperature.

4. Put these solid mediums to -20℃ fridge after sealing.

Handwriting

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image458.png)

2024.08.23 Friday

Outline:

I.Transformation:miR-142-3p without lac-operator-pET-15b plasmid
into BL21(DE3)

II.Cultivation:bacteria solution from oringinal tube [Saiyu
Luo][Huixin Liang][Rui Yao]

⑬ miR-142-3p without lac-operator-pET-15b

Detailed Experiments

I. Transformation: plasmid ⑬ to BL21

Followed the protocol : Bacterial Transformation

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image459.png)

Concentration of ⑬ : >=100ng/μL,

Heat shock at 42℃ for 90s, ice for 2 min

Add 500μL SOC medium per tube before shake for 1h

Plate transformation (200 μL) onto two 10 cm LB-Amp agar plate
containing the appropriate antibiotic.

Invert the coated plate at 37℃ and cultivate overnight.

II. Culture bacteria overnight

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image460.png)

III.Prepare SOC medium

Pack the SOC medium from 15mL centrifuge tube to 1.5mL EP
tubes(Volume: 1mL)

Figure:

Description Picture

Basic information

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image461.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image462.png)

Spread 200μL bacteria solution from the transformation on the plates

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image463.png)

SOC:Store at -80℃

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image464.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image465.png)

Handwriting

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image466.png)

2024.08.24 Saturday

Outline:

I. Transformation:

⑭ / ⑰/ ⑭+⑰ →BL21(DE3) [Saiyu Luo][Huixin Liang][Yuetong Ge]

II. Culture and store bacteria [Saiyu Luo][Huixin Liang]

III. Plate streaking ⑬ bacteria [Saiyu Luo][Huixin Liang]

IV. LB liquid/solid medium [Qiyu Tan]

V.Preparation of competent cell[Huixin Liang]

⑭miR-142-3p without lac-operator-pCDFDuet-1

⑰H210&142 without lac-operator-pCOLADuet-1

VI:Seminar of experimental group with PI Wan 【All participants】

Detailed Experiments

I. Transformation: plasmid ⑬ to BL21

Followed the protocol Bacterial Transformation

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image467.png)

Concentration of ⑭ / ⑰= 200ng/μL

Heat shock at 42℃ for 90s, ice for 2 min

Add 500μL SOC medium A/B and 1mL SOC medium C before shake for 1h

Invert the coated plate at 37℃ and cultivate overnight.

The result of plasmid ⑬ to BL21

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image468.png)

Improve: Cover the plate with sponge and filter paper which are
impregnated with RO H2O.

II. Culture bacteria overnight

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image469.png)

Stored bacteria

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image470.png)

III. Plate streaking and coating ⑬bacteria

Source: ⑬BL21(DE3)(8.23 shaking overnight) 14:00

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image471.png)

IV. LB liquid/solid medium

Follow protocol:LB liquid medium

Prepare 1.6L in total (scale errors)

Solid LB medium: 100mL K+S; 100mL K;100mL A;100mL S

V. Preparation of preparing competent cells:⑬BL21(DE3)(8.23 shaking
overnight)

Follow protocol: Preparation of competent cells

We prepare materials for the preparation of competent cells tomorrow

Weigh the drug: weigh 0.890g CaCl2

Sterilize 15mL and 50mL centrifuge tubes in one box, and sterilize
1.5mL EP tubes (more than 50 in two boxes).

Store at -20℃(1006)

Figure:

Description Picture

Plasmid information

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image472.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image473.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image474.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image475.png)

Sunday/2024.08.25

Outline:

V. Transformation:

⑬/⑮/ ⑰/⑳/㉑→BL21(DE3)/DH5a/⑦-BL21 [Saiyu Luo][Huixin Liang][Yuetong
Ge]

VI. Culture and store bacteria [Saiyu Luo][Huixin Liang][Yuetong Ge]

VII. Preparation of competent cell:⑬BL21(origin tube) [Huixin
Liang][Yuetong Ge]

VIII. Maxiprep: ⑬BL21[Qiyu Tan][Rui Yao]

IX. Preparation of LB solid medium[Qiyu Tan][Rui Yao]

⑬ miR-142-3p without lac-operator-pET-15b

⑭miR-142-3p without lac-operator-pCDF-Duet

⑮ miR-210-3p without lac-operator-pET-15b

⑰ H210&142 without lac-operator-pCOLADuet-1

⑳H01 without lac operator pCOLADuet-1-EGFP

㉑H01 without lac operator pCOLADuet-lacZa

Detailed Experiments

I. Transformation: plasmid ⑬ to BL21

Follow protocol:Bacterial Transformation

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image476.png)

Concentration of plasmid= 200ng/μL

Heat shock at 42℃ for 90s, ice for 2 min

Add 500μL SOC medium A/B and 1mL SOC medium C before shake for 1h

Invert the coated plate at 37℃ and cultivate overnight.

The result of transformation(8.24)

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image477.png)

Improve: Cover the plate with sponge and filter paper which are
impregnated with RO H2O.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image478.png)

II. Culture bacteria and store them

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image479.png)

Stored bacteria

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image480.png)

III. Preparation of competent cells:⑬BL21(DE3)(8.24 shaking
overnight)

Follow protocol:Preparation of competent cells

1.5mL glycerol/CaCl2 dissolve precipitation, 50μL per tube.(22
tubes)

Store at -80 ℃ refrigerator

Figure:

Discription Picture

Transformation

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image481.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image482.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image483.png)

Stored bacteria

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image484.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image485.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image486.png)

IV.Maxiprep: ⑬BL21

To ensure the plasmids bacteria contained is correct and get more
plasmids,we maxiprep for ⑬ from ⑬BL21 by Vigorous plasmid maxprep
kit .

Follow protocol: Plasmid Maxiprep

From step 1 to step 4, the volume of reagent added is increased in
equal proportions, multiplied from 500μL to 800μL

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image487.png)

V.Preparation of LB solid medium

preparation of LB solid medium followed the protocol

The number of each type of plate is shown in the table below

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image488.png)

Handwriting

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image489.png)

2024.08.26 Monday

Outline:

I. Store bacteria:

⑬-BL21,⑭→BL21(shake last night) [Yuetong Ge]

[20→7-BL21][17G→BL21][Huixin Liang]

II. Pick single colony and shake bacteria:

⑳→⑦-BL21,⑰→BL21,⑰+⑭→BL21[Yuetong Ge]

[20→7-BL21][13G→DH5α][15G→DH5α][21G→DH5α][Saiyu Luo]

III.Transformation:[13G][15G][20G][21G]→DH5α,[13G][15G][20G]→BL21
DE3 [Saiyu Luo][Huixin Liang][Yuetong Ge]

IV.Coat and streak plates[Huixin Liang][Rui Yao][Saiyu Luo]

V.Construct cell-free reaction system[Yuetong Ge][Sirui Dong]

VI.Cultivation:⑰G →BL21(DE3)[Huixin Liang][Rui Yao]

⑬ miR-142-3p without lac-operator-pET-15b

⑭miR-142-3p without lac-operator-pCDF-Duet

⑮ miR-210-3p without lac-operator-pET-15b⑰ H210&142 without
lac-operator-pCOLADuet-1

⑳H01 without lac operator pCOLADuet-1-EGFP

㉑H01 without lac operator
pCOLADuet-lacZaTransformation:[13G][15G][20G][21G]

Detailed Experiments

I. Store bacteria:⑬-BL21,⑭→BL21

Some of the bacteria solution shaken overnight become cloudy,so we
stored them to prepare for the further experiments.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image490.png)

Figures

Description Pictures

Store the bacteria

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image491.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image492.png)

The location of stored bacteria solution

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image493.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image494.png)

II. Shake bacteria:⑳→⑦-BL21,⑰→BL21,⑰+⑭→BL21

Some of plates from transformation in 8.25 grew,so we pick single
colonies from them and shake to prepare for cultivation and maxi
prep.

We picked single colonies from plates and shake at 37℃,220rpm

⑰+⑭→BL21 didn't become cloudy after shaking for 7.5h,we suspected
that the bacteria is not correct,so we didn't store the bacteria and
stored it at 4℃

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image495.png)

Figures

Description Pictures

Results of bacteria shaking(shake for 7.5h)

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image496.png)

Pick single colonies and shake overnight

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image497.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image498.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image499.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image500.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image501.png)

III. Transformation:[13G][15G][20G][21G]→DH5α,[13G][15G][20G]→BL21
DE3

Follow protocol: Bacterial Transformation

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image502.png)

Concentration of plasmid =200ng/μL,

Heat shock at 42℃ for 90s,ice for 2min

Add 500μL SOC medium per tube before shake for 1h

Plate transformation(200μL) onto 10cm LB agar plate containing the
appropriate antibiotic.

Invert the coated plate at 37℃ and cultivate overnight.

IV.Coat and streak plates

Some plates were suspected to have too many colonies to pick, so
they were strewn or diluted and coated.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image503.png)

Improve: Cover the plate with sponge and filter paper which are
impregnated with RO H2O.

Description Picture

Coating again

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image504.png)

streak

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image505.png)

V.Construct cell-free reaction system

1. Unfreeze all reagents (including A、B and RNase)

2. Add the reagents in order

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image506.png)

3. Mix all reagents gently in order then centrifuge the mix.

4. Incubate at 37℃ for 2h

5. Stop the reaction by ice bathing and store at -20℃

Results

97.029 mg/mL protein are measured in the mix. But no fluorescence is
observed.

VI.Cultivation:⑰G →BL21(DE3)

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image507.png)

Handwriting

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image508.png)

Tuesday /2024.08.27

Outline:

I.Transformation:⑯G→DH5α[Qiyu Tan]

II.Store bacteria [Saiyu Luo][Huixin Liang][Rui Yao][Yuetong Ge]

III.Shake bacteria [Saiyu Luo][Rui Yao]

IV.Preparation of LB medium [Qiyu Tan]

Detailed experiments

I.Transformation:⑯G→DH5α

Add 3μL plasmids into competent cells

Heat shock at 42℃for 90s

Add 500μL SOC medium per tube before shake for 1h( 220rpm, 37℃)

Spread 2 plates:

1) Spread 20μL transformation solution+180μL SOC medium mixture on
the plate.

2) Spread 200μL transformation solution on the plate.

Place the coated plate at 37℃ for 30min. After the bacterial
solution dried, invert and cultivate overnight.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image509.png)

Results(8.28 observed)

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image510.png)

II.Store bacteria

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image511.png)

Figure

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image512.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image513.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image514.png)

III.Shake bacteria

We shake the bacteria⑬G→DH5α and ⑮G→DH5α in conical flask overnight
to prepare for Maxi prep tomorrow, and pick single colonies ⑬G→BL21
from plate and shake for further experiments.

Shake at 37℃, 220rpm

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image515.png)

Figures

Description Picture

⑬G→DH5α:

8.25 transformed,

8.26 picked

shake in conical flask

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image516.png)

⑬G→DH5α:

8.25transformed,

8.27 picked

shake in conical flask

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image517.png)

⑮G→DH5α:

8.25 transformed,

8.26 picked

shake in conical flask

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image518.png)

⑮G→DH5α:

8.25transformed,

8.27 picked

shake in conical flask

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image519.png)

⑬G→Bl21(DE3):

Pick 3 single from plates and shake

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image520.png)

IV.Preparation of LB medium

Sterilization by microwave oven instead of autoclave, take LB liquid
medium which have been sterilized, and pour it into 250mL flasks,
then added agarose for 1.5g /100mL, heat for 1min in microwave,
repeat this step.

Antibiotic: Amp, 7.20, concentration = 100 mg/mL.

Handwriting

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image521.png)

Wednesday/2024.08.28

Outline:

I.Store bacteria [Qiyu Tan][Yuetong Ge]

II.Maxiprep and digestion:⑬⑮⑰BL21 [Yuxiao Qin]

III.Shake bacteria [Saiyu Luo]

IV.Transformation:⑱⑯㉒㉓⑱→DH5a[Sirui Dong][Huixin Liang]

V.IPTG induction:⑳→⑦-BL21 [Qiyu Tan]

VI.preparation of antibiotic,LB (solid) medium and plasmid[Saiyu
Luo][Huixin Liang][Qiyu Tan][Yuetong Ge]

Detailed Experiments:

I.Store bacteria

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image522.png)

Figure

Discription Picture

⑬G → BL21(DE3)

Result of shaking overnight

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image523.png)

Stored bacteria solution.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image524.png)

II.Maxiprep and digestion:⑬⑮⑰

Follow protocol:Vigorous Plasmid Maxiprep

Results

Blank concentration = 0.07 ng/μL,

Plasmid concentrations are as follow:

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image525.png)

Figures

Description Picture

Perform maxiprep on plasmid number ⑬, which is marked B on the
bottle;

Perform maxiprep on plasmid number ⑰

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image526.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image527.png)

Perform maxiprep on plasmid number ⑮, which is expanded the
cultivation in 8.27 and 8.26

Perform maxiprep on plasmid number ⑬, which is expanded the
cultivation in 8.27 and 8.26

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image528.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image529.png)

Information of the plasmids

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image530.png)

Analysis

Because the concentration was too high, we did an electrophoresis
without digesting first to see if the result was correct. Meanwhile,
the concentration of the ⑬B plasmid was unsatisfactory even after
several Vigorous Maxipreps, so we suspected that the plasmid was a
problem.

Single digestion

Follow protocol : Restriction Digest of Plasmid DNA

●We used enzyme HpaI with rCutSmartBuffer.

●We digest them at 37℃ overnight.

● The following volumes were added in the mixture:

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image531.png)

Figures

Description Picture

Digest overnight

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image532.png)

Agarose Gel Electrophoresis

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image533.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image534.png)

III.Shake bacteria

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image535.png)

Figures

Description Picture

⑳→⑦-BL21Bacteria solution came from this tube

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image536.png)

㉑G→DH5α

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image537.png)

⑭G→BL21

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image538.png)

⑰G→BL21

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image539.png)

⑰G→BL21

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image540.png)

⑳G→BL21

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image541.png)

IV.Transformation:⑱→DH5a

Follow protocol: Bacterial Transformation

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image542.png)

Concentration of plasmid= 200ng/μL

Heat shock at 42℃ for 90s, ice for 2 min

Add 500μL SOC medium before shake for 1h

Coat the Kan plate with 50μL solution and 150μL LB ,then cultivate
overnight.

Result:

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image543.png)

V. IPTG induction:⑳→⑦-BL21

Induced bacteria which has co-transformed with (20) and (7) by IPTG
for 4h, final concentration of IPTG is 0.1mM, 37℃, 180rpm.

Centrifuge bacteria solution under 8000rpm, 5min, and repeat this
step until all the solution has been centrifuged completely.

VI. Preparation of LB solid medium,antibiotic and plates pouring

Follow protocol: LB liquid medium

We prepared 2L LB in total

The concentration of antibiotic is 100μg/ml

The types of plates are as follows

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image544.png)

Preparation of Antibiotics:Amp,Kan

We prepared 5g Antibiotics(Amp ;Kan) in 50mL Centrifuge tubes

We volumed to 50mL with RO H2O

The concentration of antibiotic is 100mg/mL

To test the antibiotics,we set as follows:

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image545.png)

Dissolve plasmid

Dissolve plasmid as follows

⑱G: H210&142 pCOLADuet-1 laczα

㉒G:H142-3p 11-12 pCOLADuet-1 EGFP

㉓G:H142-3p 8-15 pCOLADuet-1 EGFP

10000g centrifuge for 1min and add 20μL ddH2O dissolve it.

Handwriting:

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image546.png)

Thursday/2024.08.29

Outline:

I.Transformation:㉔→BL21,㉖㉗㉙㉛㉜→DH5α; ㉟→BL21;⑯+⑰→⑬-BL21[Saiyu
Luo][Huixin Liang][Yuetong Ge]

II.Shake and store bacteria: ⑬→BL21; ⑭⑮F [Huixin Liang][Yuetong
Ge][Saiyu Luo]

III.Agarose gel electrophoresis: ⑬⑮⑰[Qiyu Tan]

IV.Digestion:⑬⑮⑰[Yuxiao Qin][Rui Yao]

V.Ultrasonic fragmentation of cells:⑳→⑦-BL21[Qiyu Tan]

VI.Dissolve plasmid:㉔㉖㉗㉙㉚㉛㉜㉟[Saiyu Luo]

Detailed experiments

I. Transformation:㉔→BL21,㉖㉗㉙㉛㉜→DH5α; ㉟→BL21;⑯+⑰→⑬-BL21

Followed the protocol Bacterial Transformation

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image547.png)

Concentration of plasmid= 200ng/μL

Heat shock at 42℃ for 90s, ice for 2 min

Add 500μL SOC medium before shake for 1h

Coat the Kan plate with 50μL bacteria solution and 150μL LB ,Coat
the Amp/Kan+Stc+Amp 200μL bacteria solutionwith hen cultivate
overnight.

Improve: Cover the plate with sponge and filter paper which are
impregnated with RO H2O.

Figure

Description Picture

All the plates we coated

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image548.png)

II.Shake and store bacteria: ⑬→BL21; ⑭⑮F;⑰G→DH5α;㉒G→DH5α;㉓G→DH5α

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image549.png)

Store bacteria

Shake bacteria overnight

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image550.png)

The concentration of antibiotic:100μg/ml

Figure

Disciption Picture

Pick single colonies

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image551.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image552.png)

III.Agarose gel electrophoresis: ⑬⑮⑰

Follow protocol: Agarose gel electrophoresis (unit:μL)

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image553.png)

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image554.png)

IV.Digestion:⑬⑮⑰

According to there is no marker lane on the gel,we decided to digest
the same plasmids again and run agarose gel again

Follow protocol : Restriction Digest of Plasmid DNA

●We digested them at 37℃ overnight.

●The following volumes were added in the mixture:

(Except 13B, other concentration are diluted to 1/40 of the original
concentration)

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image555.png)

P.S. “-0” represents none digestion , “-1” represents single
digestion.

V.Ultrasonic fragmentation of cells:⑳→⑦-BL21

The sample has been induced by IPTG in 8.28.

Use Ultrasonic Homogenizer SCIENTZ-IID to extract the proteins

Measured the protein concentration through nano-100.

Results

Concentration of protein samples(unit: mg/mL)

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image556.png)

Figure

Description Picture

Protein samples

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image557.png)

A,B,C are all the same, from the same bacteria solution

VI.Dissolve plasmid:㉔㉖㉗㉙㉚㉛㉜㉟

10000rpm centrifuge for 1min and add 20μL ddH2O dissolve it.

Handwriting

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image558.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image559.png)

Friday/2024.08.30

Outline:

I. The result of transformation(8.29) and single colony
picking[Huixin Liang]

II. Coat the Plate :⑯+⑰→⑬-BL21 [Huixin Liang]

III. Shake bacteria for IPTG induction:⑳→BL21[Huixin Liang]

IV.Pick single colony and shake [Sirui Dong][Yueting Guo]

V.Store bacteria[Sirui Dong][Qiyu Tan]

VI.Construct the control group of cell-free system[Yuetong Ge][Qiyu
Tan]

VII.Measure fluorescence intensity:⑳→⑦-BL21,NC- cell free,③+⑦-cell
free [Yuetong Ge][Qiyu Tan]

VIII.Agarose gel electrophoresis[Rui Yao]

⑬ miR-142-3p without lac-operator-pET-15b

⑯ miR-210-3p without lac-operator-pCDFDuet-1

⑰ H210&142 without lac-operator-pCOLADuet-1

⑳ H01 without lac operator pCOLADuet-1-EGFP

㉙Optimization 2 H210&142 pCOLADuet-1-lacZa

㉜Optimization 5 H210&142 pCOLADuet-1-lacZa

㉟ lacZw pET-15b

Detailed Experiments

I.The result of transformation and single colony picking(8.29)

Results of transformation

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image560.png)

Results of Single colony picking

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image561.png)

II.Coat the Plate :⑯+⑰→⑬-BL21

According to the results of transformation this days ,we suspected
that the plates with Kan may not be suitable for colony growth,so we
coated plates with different antibiotics to verify our conjecture.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image562.png)

Note: shake the bacteria solution for 30minutes (8.29 store at 4℃).

Results(8.31 oberved)

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image563.png)

Analysis

The two plasmids(⑯and⑰) co-transformation is unsuccessful.

II.Shake bacteria for IPTG induction:⑳→BL21

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image564.png)

Note: shake at 80rpm.

III.Pick single colony and shake

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image565.png)

IV.Store bacteria

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image566.png)

V.Construct the control group of cell-free system

The systems are as follows(unit:μL):

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image567.png)

Metal bath the positive control group at 37℃ for 2min,while store
the negative control group at -20℃ directly without metal bath after
mixing.

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image568.png)

VI.Measure fluorescence intensity:⑳→⑦-BL21,NC-cellfree,③+⑦-cellfree

Dilute the protein concentration to 1.8μg/μL

Add 100μL sample in each well

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image569.png)

VII.Agarose gel electrophoresis:⑬⑮⑰

Follow protocol: Agarose gel electrophoresis

Use 1% agarose gel

Run at 135V for 40min

The loading order and system are as follows

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image570.png)

P. S. “-0” represents none digestion , “-1” represents single
digestion.

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image571.png)

Handwriting

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image572.png)

Saturday/2024.08.31

Outline:

I. Transformation:3 groups in total

miR-142 single arm group: ⑬/⑭/- +㉒㉓㉔→BL21[Qiyu Tan][Rui Yao]

miR-210 single arm group: ⑮/⑯/- +㉕㉖㉗→BL21[Yuxiao Qin][Sirui Dong]

double arm group:⑬⑭⑮⑯/⑬+⑯/⑭+⑮/- + ⑰G[Saiyu Luo]

II.Store bacteria:⑳→BL21 [Huixin Liang][Yuetong Ge][Saiyu Luo]

III.Ultrasonic disruption of bacteria solution:㉑→DH5α [Yueting Guo]

IV.Coomassie staining of the gel:NC;PC;③+⑦ [Huixin Liang][Yuetong
Ge]

V.Preparation of plasmids [Sirui Dong][Yuxiao Qin]and LB [Qiyu
Tan][Rui Yao]

③H01 without lac operator-pET-15b(pET-15b-H01-linker-EGFP)

⑦Input 01 without lac operator-pCDFDuet-1(pCDFDuet-input01)

⑳ H01 without lac operator pCOLADuet-1-EGFP

㉑ H01 without lac operator pCOLADuet-lacZa

Detailed Experiments

I. Transformation

Follow protocol:Bacterial Transformation

miR-142 single arm group: ⑬/⑭/- +㉒㉓㉔→BL21

Dilute the plasmid as follows

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image573.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image574.png)

Mix 3μL of plasmids into 50μL of competent cells in a
microcentrifuge tube.

Put the tubes back on ice for 1 minute after heat shock.

Add 500μL LB medium (without antibiotic) after 1min ice-bath.

Spread plates for 150μL bacteria-plasmid system.

miR-210 single arm group: ⑮/⑯/- +㉕㉖㉗→BL21

Dilute the plasmid as follows

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image575.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image576.png)

Mix 3μL of plasmids into 50μL of competent cells in a
Microcentrifuge tube.

Put the tubes back on ice for 1 minute after heat shock.

Add 500μL LB medium (without antibiotic) after 1min ice-bath.

Spread plates for 150μL transformation system.

Double arm group:⑬⑭⑮⑯/⑬+⑯/⑭+⑮/- + ⑰G

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image577.png)

Concentration of plasmid=150ng/μL,2μL plasmid+50μL competent cell.

Heat shock at 42℃ for 90s,ice for 2min

Add 500mL LB medium per tube before shake for 1h

Plate transformation(150μL) onto 10cm LB agar plate containing the
appropriate antibiotic.

Invert the coated plate at 37℃ and cultivate overnight.

Figure

Description Picture

Transformation of double arm group

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image578.png)

II. Store bacteria ⑳→BL21

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image579.png)

Figure

Description Picture

Store the bacteria

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image580.png)

III. Ultrasonic disruption of bacteria ㉑→DH5a

Followed the protocol Ultrasonic disruption of bacteria

Take the supernatant and store it in three 3mL EP tubes (-80℃)and
one 1mL EP tubes(-20℃)..

IV. Coomassie staining of the gel:NC;PC;③→⑦

Follow protocol: Coomassie staining of the gel

Note:

(1)Sample Preparation

Experimental protocol: System100μL,3.6μg/μL

Heat the sample for 15 minutes at 100°C to denature the proteins.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image581.png)

INSTRUCTION MANUAL(PURExpress® In Vitro Protein Synthesis )

Loading 2.5 µl of a reaction onto a mini protein gel and resolving
by SDS-PAGE. Briefly, mix 2.5 µl of a PURExpress reaction with SDS
gel loading buffer and H2O so the total volume is 12 µL.

Heat the sample for 2 minutes at 100°C to denature the proteins and
load the sample onto a protein gel

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image582.png)

(2) The loading order and loading quantity are shown in the
following table

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image583.png)

1XBuffer: 250μL 4X sample buffer + 750μL PBS

Marker: 10μLMarker+10μL1XBuffer

(3) 90V for stacking gel ,17min ; 135v for separating gel ,40min

(4) Wash the gel with destaining solution for 15minutes, 7times.

(5) Replace the destaining solution to RO H2O and continue
decolourisation overnight

Primary result

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image584.png)

V. Preparation of plasmids and LB

Preparation of plasmids

1.Lyophilize the original tube of plasmid into a centrifuge and
centrifuge at 4℃, 10,000 xg for 1 minute.

2.Add 20μL of dd H20 to each primary tube plasmid.

Preparation of LB

Follow protocol: LB liquid medium

We totally prepared 12 plates with Amp,12 plates with Amp+Stc,12
plates with Amp+Kan

Handwriting

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image585.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj/image586.png)

2024.09.01 Sunday

Outline:

I.Coat plates[Qiyu Tan][Rui Yao][Yuxiao Qin][Sirui Dong][Yueting
Guo][Saiyu Luo]

II.Verify transformation[Sirui Dong]

III.Coomassie Brilliant blue elution [Huixin Liang][Yuetong Ge]

IV.Make competent cells:⑰→BL21,㉔→BL21 [Huixin Liang][Yuetong Ge]

V.Construct cell-free reaction system:⑳+⑦,㉟ [Huixin Liang][Yuetong
Ge]

VI.Preparation of LB medium and plates pouring [Qiyu Tan][Rui Yao]

Detailed Experiments

I.Coat plates [Qiyu Tan][Rui Yao][Yuxiao Qin][Sirui Dong][Yueting
Guo][Saiyu Luo]

Spread the bacteria solution from 8.31 transformation

The type of coated plates were as same as 8.31

Results(9.2 observed)

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image1.png)

"+" indicates that this solid medium has formed a small number of
colonies, and can pick up bacteria;

"++" indicates that this solid medium has formed a large number of
colonies and can pick up bacteria;

"-" indicates that this solid medium has not formed colonies;

"#" indicates that the bacteria have grown, but didn't form colonies
and cannot pick up bacteria;

"?" indicates that there are a few tiny dots, which couldn't affirm
that these are colonies.

Verify the resistance of plates

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image2.png)

Invert the coated plate at 37℃(in shaking table at 80 rpm) and
cultivate overnight.

Results(9.2 observed)

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image3.png)

Only the plate with Kan poured on 8.31 don't meet the expected
results,so we discard the plates poured on 8.31

Other plates meet the expected results,so we continue to use them.

II.Verify transformation[Sirui Dong]

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image4.png)

Shake at 37℃,80rpm overnight

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image5.png)

The results preliminarily prove that our transformation is
successful

III.Coomassie Brilliant blue elution [Huixin Liang][Yuetong Ge]

Repeat the elution with the eluent three times for 15min each time.

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image6.png)

Analysis

1.Positive control show the band at about 20kD(about the right
size),which means the PURE system is effective and it can express
DHFR

2.③+⑦ group didn't show any band more than nagative control,to find
the problem,we decided to construct cell free system with
plasmid⑳+⑦.

IV.Make competent cells:⑰→BL21,㉔→BL21 [Huixin Liang][Yuetong Ge]

We shook ⑰→BL21,㉔→BL21,⑮-BL21,⑯-BL21 to make competent cells,but
⑮-BL21,⑯-BL21 didn't grow after shaking for 6h,so we only make
⑰→BL21,㉔→BL21 competent cells

Follow protocol:Preparation of competent cells

Add 1ml bacteria solution from 15ml tube stored at 4℃ to 200ml LB
with antibiotic(concentration:100μg/ml) before making

We only use 100ml of bacteria solution per group to prepare
competent cells

Figures

description Picture

shake 4 groups of bacteria to prepare for competent cells making

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image7.png)

The bacteria solution added to 200ml LB

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image8.png)

V.Construct cell-free reaction system:⑳+⑦,㉟ [Huixin Liang][Yuetong
Ge]

The systems are as follows(unit:μL):

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image9.png)

Store the solution at -20℃ after reaction

VI.Preparation of LB medium and plates pouring [Qiyu Tan][Rui Yao]

Follow protocol:LB liquid medium

Prepare 4 bottles for LB solid medium, 5 bottles for LB liquid
medium in total

Pour 8 plates with Kan,11 plates with Amp+Stc,12 plates with Amp+Kan
and 12 plates with Kan+Stc

2024.09.02 Saturday

Outline:

I. Detection of fluorescence[Yuetong Ge][Qiyu Tan]

II. CPRG color reaction [Huixin Liang][Yuetong Ge]

III. Single Colony Pick[Saiyu Luo][Huixin Liang][Yuetong Ge]

IV. Vigorous Maxiprep and digestion : ⑬A
;⑯(50+150);⑯(100)⑮9.1-I;⑮9.1-II;⑰BL21 [Yuxiao Qin][Rui Yao] [Yuetong
Ge]

Detailed Experiments

I.Detection of fluorescence[Yuetong Ge][Qiyu Tan]

Add 100μL sample to each well

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image10.png)

II.CPRG color reaction [Huixin Liang][Yuetong Ge]

We design the system followed by the technical@GBiosciences.com

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image11.png)

Note:

Prepare 25X CPRG Substrate by adding 550µl of CPRG Assay Reaction
Buffer into a vial of CPRG Substrate. To completely dissolve the
substrate, mix the vial by vortexing for 20 seconds.

β-gal(0.05μg/μL):Weigh the 0.01g powder with 20mL (1X pH=7.3)PBS,
dispense in 1.5 mL EP tubes.(Store at -20℃)

The result is satisfactory, the reaction is quick, the sample turns
dark red immediately.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image12.png)

III.Pick Single Colony [Huixin Liang][Yuetong Ge][Saiyu Luo]

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image13.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image14.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image15.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image16.png)

IV.Vigorous Maxiprep and digestion : ⑬A
;⑯(50+150);⑯(100)⑮9.1-I;⑮9.1-II;⑰BL21 [Yuxiao Qin][Rui Yao] [Yuetong
Ge]

Follow protocol:Vigorous Plasmid Maxiprep

Results

Blank concentration:0.042ng/μL

Plasmid concentration were as follows(unit:ng/μL):

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image17.png)

Follow protocol:Restriction Digest of Plasmid DNA

●We digest them at 37℃ for 1h.

●The following volumes were added in the mixture(units:μL):

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image18.png)

Handwriting

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image19.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image20.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image21.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image22.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image23.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image24.png)

2024.09.03 Monday

Outline:

I.Transformation:⑬+㉓,⑬+㉔,⑭+㉒,⑭+㉓[Qiyu Tan][Rui Yao]

II.Store bacteria [Huixin Liang]

III.Agarose gel electrophoresis[Yueting Guo]

IV.Preparation of LB medium[Yueting Guo]

Detailed Experiment:

I.Transformation:⑬+㉓,⑬+㉔,⑭+㉒,⑭+㉓[Qiyu Tan][Rui Yao]

Follow protocol:Transformation

Dilute the plasmid first

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image25.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image26.png)

Put the tubes back on ice for 1 minute after heat shock.

Add 500μL LB medium (without antibiotic) after 1min ice-bath.

Spread 150μL bacteria solution on plates

Incubate plates in 37 °C Shaking incubator overnight

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image27.png)

II.Store bacteria [Huixin Liang][Saiyu Luo]

All the bacteria shaken yesterday(9.2,overnight) grew well,so we
store them and store the rest of bacteria solution at 4℃

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image28.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image29.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image30.png)

Corresponding 15mL tubes in order:

Line 1:

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image31.png)

Line 2:

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image32.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image33.png)

Line 3/4/5:

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image34.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image35.png)

III.Agarose gel electrophoresis[Yueting Guo]

Follow protocol:Agarose gel electrophoresis

Use 1% agarose gel

Run at 135V for 40min

The loading order and system are as follows(unit:μL)

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image36.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image37.png)

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image38.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image39.png)

-0 means it's not digested by enzyme

-1 means it's digested by single enzyme

Analysis

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image40.png)

Incorrect bands appeared in the 16-II-single digestion may caused by
the unfinished digestion

The reason why 15-I-NULL couldn't see clear bands was still unknown

The reason why 15-I-single digestion has totally wrong bands was
still unknown

Sequencing results

⑬A,⑮II,⑯I are right

⑬A

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image41.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image42.png)

⑮II

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image43.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image44.png)

⑯I

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image45.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image46.png)

IV.Preparation of LB medium[Yueting Guo]

Follow protocol:LB liquid medium

We prepare 2.5L LB in total

Handwriting

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image47.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image48.png)

2024.09.04 Wednesday

Outline:

I.Shake the bacteria[Qiyu Tan]

II.Store the bacteria:⑬+㉓,⑬+㉔,⑭+㉒[Rui Yao]

III.IPTG induction[Sirui Dong]

IV.Coat the plates[Qiyu Tan]

Detailed Experiments:

I.Shake the bacteria[Qiyu Tan]

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image49.png)

II.Store the bacteria:⑬+㉓,⑬+㉔,⑭+㉒[Rui Yao]

All the bacteria shaken tomorrow grew well,so we store them at
-80℃and store the rest of bacteria solution at 4℃.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image50.png)

Figures

Note Pictures

Strain preservation

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image51.png)

Location of stored bacteria

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image52.png)

Location of rest of bacteria

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image53.png)

III.IPTG induction[Sirui Dong]

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image54.png)

After induction,we centrifuge the bacteria solution at 8000rpm,5min
each time and store the precipitation at -80℃

IV.Coat the plates:⑭+㉓[Qiyu Tan]

Spread 150μL bacteria solution from 9.3 transformation on plates and
incubate at 37℃

Handwriting

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image55.png)

2024.09.05 Thursday

Outline:

I. Pick single colony:⑭+㉓G[Qiyu Tan]

II. Preparation of IPTG[Huixin Liang][Yuetong Ge]

III.Store bacteria:⑭+㉓G [Saiyu Luo]

IV.IPTG induction [Saiyu Luo]

V.Construct cellfree system:positive control,③+⑦(IPTG+/-) [Huixin
Liang][Yuetong Ge]

VI.SDS-PAGE:NC(8.30),PC(8.30),PC(9.5),③+⑦(IPTG+/-)(9.5)[Huixin
Liang]

VII.Ultrasonic disruption of bacteria:⑯+㉕.⑯+㉖,⑯+㉗,㉕[Yueting Guo]

VIII.Preparation of LB medium[Yueting Guo]

Detailed experiments

I.Pick single colony and shake bacteria:⑭+㉓G[Qiyu Tan]

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image56.png)

II.Preparation of IPTG [Huixin Liang][Yuetong Ge]

We prepare 10ml IPTG (concentration=0.5M)in total

We use UP H2O to dissolve IPTG

III.Store bacteria:⑭+㉓G [Saiyu Luo]

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image57.png)

IV.IPTG induction, [Saiyu Luo]

Shake for 4h before adding IPTG

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image58.png)

Add 40μL IPTG(c=0.5M) to cultured bacteria solution and shake for
another 4h

Centrifuge the bacteria solution at 5000rpm for 10min after IPTG
induction and store the precipitation at -80℃

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image59.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image60.png)

V.Construct cellfree system and fluorescence measurement:positive
control,③+⑦(IPTG+/-) [Huixin Liang][Yuetong Ge]

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image61.png)

Incubate them at 37℃ for 6h

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image62.png)

Fluorescence detection

Dilute the samples to 1.8μg/ul or 9μg/μL

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image63.png)

Analysis

IPTG induction and prolonged reaction time did not help protein
expression

VI.SDS-PAGE:NC(8.30),PC(8.30),PC(9.5),③+⑦(IPTG+/-)(9.5)[Huixin
Liang]

We forgot to lane marker so we decided to do it again tomorrow

VII.Ultrasonic disruption of bacteria:⑯+㉕.⑯+㉖,⑯+㉗,㉕.[Yueting
Guo]

Follow protocol:Ultrasonic disruption of bacteria

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image64.png)

VIII.Preparation of LB medium[Yueting Guo]

Follow protocol:LB liquid medium

We prepare 2L LB in total

Handwriting

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image65.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image66.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image67.png)

2024.09.06 Friday

Outline:

I.Ultrasonic disruption of bacteria and fluorescence measurement:
⑬+⑰,⑯+⑰,⑬+⑯+⑰[Saiyu Luo][Qiyu Tan]

II.IPTG induction [Sirui Dong][Yuxiao Qin]

III.Store bacteria[Sirui Dong][Yuxiao Qin]

IV.Construct the cell-free system:⑦+㉑+㉟; ㉞+⑦ [Huixin
Liang][Yuetong Ge]

V.Coomassie staining of the gel:NC;PC1/2;㉟;③→⑦ IPTG +/-[Huixin
Liang][Yuetong Ge]

Detailed experiments

I.Ultrasonic disruption of bacteria and Measure
fluorescence:⑬+⑰→BL21,⑯+⑰→BL21,⑬+⑯+⑰→BL21 [Saiyu Luo][Qiyu Tan]

Ultrasonic disruption of bacteria

Follow protocol: Ultrasonic disruption of bacteria

Results(unit:mg/ml)

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image68.png)

Fluorescence Measurement

Dilute the samples to 1.8μg/ul

Add 100μL sample to each hole

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image69.png)

Figure

description Picture

Centrifuge the bacteria solution

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image70.png)

Get the solution of lysis

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image71.png)

Measure the fluorescence

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image72.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image73.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image74.png)

II.IPTG induction [Sirui Dong][Yuxiao Qin]

Add 40μL IPTG(c=0.5M) to bacteria solution as follows:

⑬+㉒,⑬+㉓,⑬+㉔,⑭+㉒fail to grow,so we plan to do it one more time.

We shook ⑯+㉕,⑯+㉖,⑯+㉗,㉖,㉗ again because of mistake

Shake for another 4h at 220rpm,37℃

III.Store bacteria[Sirui Dong][Yuxiao Qin]

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image75.png)

IV.Construct the cell-free system:⑦+㉑+㉟; ㉞+⑦ [Huixin
Liang][Yuetong Ge]

The systems are as follows(unit:μL):

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image76.png)

The concentration of Plasmid:㉑/㉟/㉞=200ng/μL, ⑦=937ng/μL

Metal bath at 37 ° C for 6h

V.Coomassie staining of the gel:NC;PC;㉟;③→⑦ [Huixin Liang][Yuetong
Ge]

(1)INSTRUCTION MANUAL(PURExpress® In Vitro Protein Synthesis )

Loading 2.5 µl of a reaction onto a mini protein gel and resolving
by SDS-PAGE. Briefly, mix 2.5 µl of a PURExpress reaction with SDS
gel loading buffer and H2O so the total volume is 12 µL.

Heat the sample for 2 minutes at 100°C to denature the proteins and
load the sample onto a protein gel

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image77.png)

(2) The loading order and loading quantity are shown in the
following table

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image78.png)

1XBuffer: 250μL 4X sample buffer + 750μL PBS

Marker: 10μLMarker+10μL1XBuffer

(3) 90V for stacking gel ,20min ; 135v for separating gel ,40min

(4) Wash the gel with destaining solution for 15minutes,2times

(5) Replace the destaining solution to RO H2O and continue
decolourisation overnight

Figure

Description Picture

Make the sample of SDS-PAGE

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image79.png)

Handwriting

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image80.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image81.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image82.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image83.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image84.png)

2024.09.07 Saturday

Outline:

I.IPTG induction:⑰G→BL21(DE3) [Saiyu Luo]

II.Pick Single Colony [Saiyu Luo]

III.Coomassie Brilliant blue elution[Huixin Liang][Yuetong Ge]

IV.Preparation of LB[Yuxiao Qin][Rui Yao][Sirui Dong]

Detailed Experiments:

I. IPTG induction:⑰G→BL21(DE3)[Saiyu Luo]

Add 40μL IPTG to 200ml bacteria solution and shake for another 4h

Centrifuge the bacteria solution and store precipitation at -80℃

II. Single Colony Pick[Saiyu Luo]

Pick Single Colony as follows:

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image85.png)

Figure

Description Picture

Picked the single colony and shook overnight at 37℃

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image86.png)

III. Coomassie blue Eluent configuration and elution for Coomassie
brilliant blue staining [Huixin Liang][Yuetong Ge]

Coomassie blue Eluent configuration

Follow protocol:Destaining solution

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image87.png)

Mix well and store at room temperature

Coomassie Brilliant blue elution[Huixin Liang][Yuetong Ge]

Follow protocol:Coomassie staining of the gel

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image88.png)

IV.Preparation of LB[Yuxiao Qin][Rui Yao][Sirui Dong]

Followed the protocol:LB liquid medium

We prepare 2.4L LB in total

We use ⑯+⑰→BL21 to verify the LB medium is suitable for bacterial
growth

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image89.png)

Handwriting

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image90.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image91.png)

2024.09.08 Sunday

Outline:

I.Ultrasonic disruption of bacteria and measurement of
flurencense:⑰G→BL21(DE3) [Saiyu Luo]

II.IPTG
induction:⑬+㉒G,⑬+㉓G,⑬+㉔G,⑭+㉒G,⑮+㉕G,⑮+㉖G,⑮+㉗G,㉖G,㉗G→BL21
[Saiyu Luo][Qiyu Tan]

III.Pick single colony and shake:⑬+㉒G[Qiyu Tan]

IV.Shake bacteria[Qiyu Tan]

Detailed experiments

I.Ultrasonic disruption of bacteria and measurement of
flurencense:⑰G→BL21(DE3) [Saiyu Luo]

Ultrasonic disruption of bacteria

Follow protocol:Ultrasonic disruption of bacteria

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image92.png)

Measurement of flurencense

Follow protocol:Multi-Mode Microplate Reader

Dilute the sample to 1.8 μg/μL, total volume=100μL

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image93.png)

Figure

Description Picture

Result of flurencense measureing

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image94.png)

II.IPTG
induction:⑬+㉒G,⑬+㉓G,⑬+㉔G,⑭+㉒G,⑮+㉕G,⑮+㉖G,⑮+㉗G,㉖G,㉗G→BL21

Shake at 220rpm for 4h before adding IPTG

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image95.png)

Add 40μL IPTG(c=0.5M) to 200ml bacteria solution and shake for
another 4h

Centrifuge the bacteria solution at 5000rpm for 10min,and store the
precipitation at -80℃

Figure

description Picture

The result of shaking for 4h before adding IPTG

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image96.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image97.png)

Centrifuge the bacteria solution after IPTG induction

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image98.png)

III.Pick single colony and shake[Qiyu Tan]

Because we failed to cultivate the bacteria in IPTG induction,we
picked single colonies again and shook to prepare for the
experiments tomorrow.

Pick 3 single colonies from the plates coated on 9.1,add 5ml LB and
shake at 220rpm overnight

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image99.png)

IV.Shake bacteria[Qiyu Tan]

To prepare for the maxiprep, we shake bacteria in the conical flask
overnight.

Handwriting

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image100.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image101.png)

2024.09.09 Monday

Outline:

I.Construct cell-free reaction system:㉟ [Yuetong Ge]

II.Make sample of SDS-PAGE:㉟(cellfree),⑳→⑦-BL21,㉑→DH5α[Yuetong Ge]

III.Store bacteria[Yuetong Ge][Sirui Dong]

IV.Transformation:㉘/⑬+㉘/⑯+㉘/⑬+⑯+㉘→BL21[Saiyu Luo]

V.Ultrasonic disruption of bacteria:㉖,⑮+㉕,⑮+㉖,⑮+㉗[Sirui Dong]

VI.Vigorous Maxiprep:⑬+⑰,⑯+⑰,⑬+⑯+⑰[Qiyu Tan]

VII.Shake bacteria:Group of miR-210[Yuxiao Qin]

Detailed Experiments

I.Construct cell-free reaction system:㉟ [Yuetong Ge]

unit:μL

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image102.png)

Incubate at 37℃ for 6h

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image103.png)

II.Make sample of SDS-PAGE:㉟(cellfree),⑳→⑦-BL21,㉑→DH5α[Yuetong Ge]

System(unit:μL)

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image104.png)

Heated the sample at 100℃ for 3min and stored at -20℃ for SDS-PAGE
tomorrow

III.Store bacteria[Yuetong Ge][Sirui Dong]

Follow protocol:Strain preservation

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image105.png)

IV.Transformation:㉘/⑬+㉘/⑯+㉘/⑬+⑯+㉘→BL21[Saiyu Luo]

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image106.png)

Heat shock at 42℃ for 90s,on ice for 2min

Add 500μL LB medium per tube before shake for 1h

Plate transformation(200μL) onto 10cm LB agar plate containing the
appropriate antibiotic. (400μL for 13G+16G+28G)

Invert the coated plate at 37℃ overnight.

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image107.png)

Figure

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image108.png)

Description Picture

The plasmid used for transformation

V.Ultrasonic disruption of bacteria and measurement of
flurencense:㉖,⑮+㉕,⑮+㉖,⑮+㉗[Sirui Dong]

Ultrasonic disruption of bacteria

Follow protocol:Ultrasonic disruption of bacteria

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image109.png)

VI.Vigorous Maxiprep:⑬+⑰,⑯+⑰,⑬+⑯+⑰

Follow protocol:Vigorous Plasmid Maxiprep

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image110.png)

VII.Shake bacteria:group of miR-210

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image111.png)

Handwriting

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image112.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image113.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image114.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image115.png)

2024.09.10 Tuesday

Outline:

I.SDS-PAGE and coomassie bright blue staining of the
gel:⑳→⑦-BL21,㉑→DH5α,㉟(cellfree) [Huixin Liang]

II.Single colony pick:㉘→BL21,⑬+㉘→BL21 ,⑯+㉘→BL21[Saiyu Luo]

III.Shake the bacteria:⑬+㉓→BL21,⑬+㉔→BL21, ⑭+㉒→BL21,⑭+㉓→BL21[Qiyu
Tan]

IV.Preparation of LB medium [Qiyu Tan]

V.Vigorous maxirprep:group of miR-210[Yuxiao Qin]

Detailed Experiments

I.SDS-PAGE and coomassie bright blue staining of the
gel:⑳→⑦-BL21,㉑→DH5α,㉟(cellfree) [Huixin Liang]

Follow protocol:Western Blot and Commassie staining of the gel

SDS-PAGE

The loading order and loading quantity are shown in the following
table

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image116.png)

1XBuffer: 250μL 4X sample buffer + 750μL PBS

Marker: 10μLMarker+10μL1XBuffer

90V for stacking gel ,20min ; 135v for separating gel ,50min

Coomassie bright blue staining of the gel

Stain the gel with coomassie briliant blue staining solution for
40min

Wash the gel with destaining solution for 15minutes,2times

Replace the destaining solution to RO H2O and continue
decolourisation overnight

Figure

description Picture

The gel after staining

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image117.png)

II.Single colony pick [Saiyu Luo]

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image118.png)

Shake overnight at 220rpm,37℃

Figure

description Picture

Shake bacteria in 15ml tube

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image119.png)

III.Shake the bacteria:⑬+㉓→BL21,⑬+㉔→BL21, ⑭+㉒→BL21,⑭+㉓→BL21[Qiyu
Tan]

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image120.png)

IV.Preparation of LB medium [Qiyu Tan]

Follow protocol:LB liquid medium

We prepare 2L LB in total

V.Vigorous maxirprep:group of miR-210[Yuxiao Qin]

Follow protocol:Vigorous Plasmid maxiprep

Results

Blank=0.061ng/μL

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image121.png)

Figure

Description Picture

The plasmid we extracted

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image122.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image123.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image124.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image125.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image126.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image127.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image128.png)

2024.09.11 Wednesday

Outline:

I.Primary part of WB:⑳→⑦-BL21,㉑→DH5α,㉟(cellfree) [Huixin
Liang][Yuetong Ge][Sirui Dong] [Qiyu Tan]

II.The result of Coomassie bright blue
dye:⑳→⑦-BL21,㉑→DH5α,㉟(cellfree)[Huixin Liang][Yuetong Ge]

III.Store the bacteria[Qiyu Tan]

IV.Shake the bacteria and IPTG induction[Qiyu Tan]

V.Digestion and Agarose gel electrophoresis:group of miR-210[Yuxiao
Qin][Qyu Tan]

Detailed Experiments

I.Primary part of WB:⑳→⑦-BL21,㉑→DH5α,㉟(cellfree)[Yuetong Ge]
[Huixin Liang]

Followed the protocol:Western Blot

The loading order and system are shown in the following table

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image129.png)

Run at 80V for stacking gel ,20min ;135v for separating gel ,45min

Prepration of milk and antibody [Sirui Dong] [Qiyu Tan]

Followed the protocol: Milk(5%) Primary antibody Secondary antibody

II.The result of Coomassie bright blue
dye:⑳→⑦-BL21,㉑→DH5α,㉟(cellfree)

[Yuetong Ge] [Huixin Liang]

There is no visible band of β-gal on the gel.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image130.png)

III.Store the bacteria[Qiyu Tan][Saiyu Luo]

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image131.png)

IV.Shake the bacteria and IPTG induction[Qiyu Tan][Sirui Dong]

Shake before IPTG induction

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image132.png)

Add 40μL IPTG (c=0.5M) to each conical flask and shake for another
4h

Centrifuge the bacteria solution at 5000rpm for 10min each time

Store the precipitation at -80℃

Shake in 15ml tubes

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image133.png)

Shake before maxiprep

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image134.png)

V.Digestion and Agarose gel electrophoresis:group of miR-210[Yuxiao
Qin][Qyu Tan]

Follow protocol:Restriction Digest of Plasmid DNA Agarose gel
electrophoresis

Digest at 37℃for 75min

The system of digestion is as follows

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image135.png)

Use 1% agarose gel

Run at 135V for 40min

The loading sequence and systems are shown in the following table

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image136.png)

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image137.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image138.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image139.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image140.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image141.png)

2024.09.12 Thursday

Outline:

I. Subsequent part of WB:⑳→⑦-BL21,㉑→DH5α,㉟(cellfree)[Yueting
Guo][Yuetong Ge]

II.Make sample of
SDS-PAGE:㉟(cellfree),⑳→⑦-BL21(cellfree);⑳→⑦-BL21,㉑→DH5α;

[Huixin Liang] [Yuetong Ge]

III.Transformation :⑯+㉘→⑬-BL21(competent cell) [Saiyu Luo][Huixin
Liang]

IV.Vigorous maxiprep and digestion[Yuxiao Qin][Rui Yao][Yuetong Ge]

V.Ultrasonic disruption of bacteria [Qiyu Tan]

VI.IPTG induction[Qiyu Tan]

VII.Preparation of LB medium[Qiyu Tan]

Detailed Experiments

I.Subsequent part of WB:⑳→⑦-BL21,㉑→DH5α,㉟(cellfree)[Yuetong
Ge][Yueting Guo]

Follow protocol:Western Blot

Results

112-116kD has light bands showing LacZω expression, but lacks a
negative control only contain conponents of cell-free system, so we
need to improve our experimental scheme.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image142.png)

II.Make sample of
SDS-PAGE:㉟(cellfree),⑳→⑦-BL21(cellfree);⑳→⑦-BL21,㉑→DH5α;

[Yuetong Ge] [Huixin Liang]

Follow protocol:Western Blot

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image143.png)

Heat the sample at 100℃ for 15min and store at -20℃ for WB tomorrow

III.Transformation:⑯+㉘→⑬-BL21,㉛/⑬+㉛/⑯+㉛→BL21[Saiyu Luo][Huixin
Liang]

⑯+㉘→⑬-BL21[Saiyu Luo][Huixin Liang]

Follow protocol:Transformation

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image144.png)

The concentration of the plasmid㉘=200ng/μL

Heat-shock at 42℃ for 90s

Add 500μL LB medium per tube before shake for 1h

Place the coated plate under 37℃ for 30min,invert and cultivate
overnight

Transformation:㉛/⑬+㉛/⑯+㉛→BL21

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image145.png)

Heat shock at 42℃ for 90s,on ice for 2min

Add 500μL LB medium per tube before shaking for 1h

Spread bacteria solution from transformation(200μL) on 10cm LB agar
plate containing the appropriate antibiotic.

Invert the coated plate and cultivate at 37℃overnight.

IV.Vigorous maxiprep and digestion[Yuxiao Qin][Rui Yao][Yueton Ge]

Maxiprep

Follow protocol:Vigorous plasmid maxiprep

Results

blank concentration=0.009 ng/μL

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image146.png)

Follow protocol:Restriction Digest of plasmid DNA

We used enzyme HpaI with rCutSmart Buffer.

We digest them at 37℃for 1h(A portion of the plasmid was digested at
37 degrees for one hour)

The system of digestion is as follows:

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image147.png)

V.Ultrasonic disruption of bacteria [Qiyu Tan]

Follow protocol:Ultrasonic disruption of bacteria

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image148.png)

VI.IPTG induction:㉘,⑬+㉘,⑯+㉘→BL21[Qiyu Tan]

Shake 4h before adding IPTG

Add 40μL IPTG(c=0.4M) to each group and shake for another 4h

VII.Preparation of LB medium[Qiyu Tan]

Follow protocol:LB liquid medium

We prepare 2.4L LB in total

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image149.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image150.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image151.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image152.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image153.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image154.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image155.png)

2024.09.13 Friday

Outline:

I. Primary part of WB:
⑳→⑦-BL21,㉑→DH5α,㉟(cellfree);⑳→⑦-BL21(cellfree)

[Huixin Liang][Yuetong Ge]

II.Single Colony Pick [Saiyu Luo][Yuetong Ge]

III.Agarose Gel Electrophoresis [Saiyu Luo][Yuetong Ge]

IV.Plates pouring and preparation of Running buffer [Yuetong Ge]

Detailed Experiments:

I.Primary part of
WB:⑳→⑦-BL21,㉑→DH5α,㉟(cellfree),⑳→⑦-BL21(cellfree) [Yuetong
Ge][Huixin Liang]

Followed the protocol:Western Blot

The loading order and system are shown in the following table

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image156.png)

80V for stacking gel, 15min ;

135v for separating gel , 50min

Primary antibody:(1)-His-tag;(2)-LacZ polyclonal antibody

II.Single Colony Pick [Saiyu Luo][Yuetong Ge]

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image157.png)

Shake overnight at 220rpm,37℃

Figure

Description Picture

Pick the single colony from these plates

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image158.png)

III.Agarose Gel Electrophoresis [Saiyu Luo][Yuetong Ge]

Follow protocol:Agarose Gel Electrophoresis

Use 1% agarose gel

4μL 6xloading buffer was added in each sample

Run the gel at 135V for 40min

The loading order and system are shown in the following
table(unit:μL)

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image159.png)

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image160.png)

IV.Plates pouring and preparation of Running buffer [Yuetong Ge]

Follow protocol:LB liquid medium and Running buffer(10x)

We pour 3 types of plates:Kan+Amp+Stc,Amp+Stc,Amp

We prepare 1L Running buffer in total

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image161.png)

2024.09.14 Saturday

Outline:

I.Transformation:⑬+⑯+㉘→BL21,⑬+⑯+㉛→BL21 [Saiyu Luo]

II.Ultrasonic disruption of bacteria and Measurement by Multi-mode
Microplate Reader [Saiyu Luo]

III.Shake bacteria[Rui Yao]

IV.Subsequent part of WB:⑳→⑦-BL21,㉑→DH5α,㉟(cellfree),⑳→⑦(cellfree)
[Yueting Guo][Yuetong Ge]

Detailed experiments:

I.Transformation[Saiyu Luo]

Heat shock at 42℃ for 90s,on ice for 2min

Add 500μL LB medium per tube before shake for 1h

Plate transformation(200μL) onto 10cm LB agar plate containing the
appropriate antibiotic.

Invert the coated plate at 37℃ and cultivate overnight.

Plasmid concentration are as follows

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image162.png)

System of transformation

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image163.png)

II.Ultrasonic disruption of bacteria and Measurement by Multi-mode
Microplate Reader [Saiyu Luo]

Ultrasonic disruption of bacteria

Follow protocol:Ultrasonic disruption of bacteria

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image164.png)

Fluorescence measurement

Dilute all protein samples to 1.8 μg/μL by PBS, and add 100μL in
each hole to measure EGFP fluorescence.

PMT Gain:Medium High

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image165.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image166.png)

Figure

description Picture

Get the bacterial lysate

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image167.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image168.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image169.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image170.png)

Measure the fluorescence

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image171.png)

III.Shake bacteria[Rui Yao]

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image172.png)

Shake at 220rpm,37℃

Figure

description Picture

Add antibiotic to LB medium

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image173.png)

Shake the bacteria in 15ml tube

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image174.png)

IV.Subsequent part of WB:⑳→⑦-BL21,㉑→DH5α,㉟(cellfree),⑳→⑦(cellfree)
[Yueting Guo][Yuetong Ge]

Follow protocol:Western Blot

Results

Primary antibody:LacZ Polyclonal antibody

112-116kD has light bands showing LacZω expression, but the negative
control only containing conponents of cell-free system ,expresses
the same.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image175.png)

Primary antibody:His-tag antibody

No specific LacZω-expressing bands were found

Negative control had multiple bands expressing with LacZω

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image176.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image177.png)

Sunday/2024.09.15

Outline:

I.Construct cell-free system:㉟; ④;PC [Huixin Liang][Yuetong Ge]

II.Coomassie staining of the gel:㉟; ④;PC [Huixin Liang][Yuetong Ge]

III.Trasformation:㉒,㉓,㉔,⑬+㉒→BL21[Qiyu Tan][Rui Yao][Sirui Dong]

IV.Preparation of LB medium[Qiyu Tan][Rui Yao]

V.Coat Plate:⑬+⑯+㉘→BL21[Qiyu Tan]

Detailed Experiments:

I. Construct the control group of cell-free system

The systems are as follows(unit:μL):

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image178.png)

The concentration of plasmid:㉟=200ng/μL, ④=950ng/μL

Metal bath at 37 ° C for 5h

II. Coomassie staining of the gel:㉟; ④;PC

Follow protocol:Coomassie staining of the gel

Sample Preparation

Experimental protocol: System48μL,9μg/μL

Heat the sample for 15 minutes at 100°C to denature the proteins.

The loading order and system are as follows:

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image179.png)

III.Trasformation:㉒,㉓,㉔,⑬+㉒→BL21[Qiyu Tan][Rui Yao][Sirui Dong]

Follow Protocol:Bacterial Transformation

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image180.png)

The concentration of plasmid is 100ng/μL

Put the tubes back on ice for 1 minute after heat shock.

Add 500μL LB medium (without antibiotic) after 1min ice-bath.

IV.Preparation of LB medium[Qiyu Tan][Rui Yao]

Follow protocol:LB liquid medium

We prepare 1.4L LB in total

V.Coat Plate:⑬+⑯+㉘→BL21[Qiyu Tan]

We use bacteria solution from 9.14 transformation to coat plates
again

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image181.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image182.png)

2024.09.16 Monday

Outline:

I. Primary part of WB:㉟; ④;PC [Huixin Liang][Yuetong Ge]

II. RNA extraction in vitro ⑦;⒂-II [Huixin Liang][Yuetong Ge]

III.Shake bacteria and IPTG induction [Sirui Dong][Qiyu Tan][Rui
Yao][Saiyu Luo]

IV.Preparation of LB medium [Rui Yao][Qiyu Tan]

V.Shake bacteria:group of miR-142[Qiyu Tan][Rui Yao]

Detailed Experiments

I.Primary part of WB:㉟(cellfree) ; ④(cellfree) ;PC(cellfree)
[Yuetong Ge][Yueting Guo]

Follow the protocol:Western Blot

The loading order and systems are shown in the following table

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image183.png)

50μL Marker configuration :25μL Marker+25μL 1xBuffer

80V for stacking gel, 20min

135v for separating gel , 50min

Primary antibody:(1)-His-tag;(2)-LacZ polyclonal antibody

II. RNA extraction in vitro

1. DNA template preparation:Single enzyme digestion

In order to obtain a specific length RNA, the plasmid must be
completely linearized.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image184.png)

Digest at 37℃ for 2h

⑦: Split into five systems(20 μL per system)

2. In vitro RNA transcription

(1)Thawing reagents:

Centrifuge the T7 RNA Polymerase Mix briefly and place on ice. Thaw
10× Transcription Buffer and ribonucleotides (ATP, CTP, GTP, UTP),
mix and centrifuge to the bottom of the tube, place 10×
Transcription Buffer at room temperature, and place 4 types of
ribonucleotides on ice.

(2) Assembly transcription reaction at room temperature

Prepare the reaction system according to the following system:

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image185.png)

Note:

1. The reaction is configured at room temperature. Since 10×
Transcription Buffer contains spermidine, the concentration of
spermidine too high will cause DNA template precipitation at low
temperature.

2. Short transcript (<100 nt), 2 µg template can be used,
transcription time increased to 4-8 hs.

3. For long transcripts (>1000 nt), recommended to use linearized
plasmid templates for transcription.

4. Perform the reaction in a PCR machine with the hot lid open to
prevent the reaction solution from evaporating for a long time.

5. The reaction product may have a white precipitate. This is free
pyrophosphate and magnesium ions produce the magnesium

pyrophosphate in the reaction, won't affect the subsequent
experiments. You can add some EDTA to clear it. If the addition of

EDTA affects subsequent experiments, the supernatant can also be
recovered by centrifugation.

6. The reagents and containers must without RNase contamination.

(3) Incubate at 37°C for 4 hours

(4) Before purification, add 80μL RNase free H2O to dilute the
product to 100 μL,

3. Product purification

Follow protocol: EasyPure® RNA Kit

(1) Measure the volume of the flow-through accurately andtransfer it
to a clean 1.5 ml RNase-free centrifuge tube. Add 1.25 volumes
(125μL)of absolute ethanol to the tube .Some precipitates may appear
at this moment. Invert the tubegently to mix well.

(2) Add all the solution and precipitates into the miRNA Spn Column.
Centrifuge at 12,000xg for 30 seconds at roomtemperature, and
discard the flow-through (If the volumof the mixture is larger than
the maximum sample volume thecolumn can process, repeat this step
until all the mixturhas been loaded)

(3) Add 500 μL ofWB10 (check to make sure that absolutethanol has
been added prior to use) into the spin column. Centrifugethe
flow-through.at 12,000xg for 30 seconds at room temperature. Discar

(4) Repeat step(3)once.

(5) Centrifuge at 12,000 xg for 2 minutes at room temperatre to
remove ethanol residue thoroughly.

(6) Place the miRNA spin column into a 1.5 mL RNase-freetube. Add 30
μL of RNase-free Water to the center of the spincolumn matrix and
incubate at room temperature for 1 nlinute.

(7) Centrifuge at 12,000xg for 1 minute to elute miRNA.

(8) Store the isolated miRNA at -80°C

4. RNA quantification

The concentration of RNA:

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image186.png)

5. RNA size and quality detection

Agarose electrophoresis

In order to determine the size, integrity and quality of RNA,
agarose gel electrophoresis or polyacrylamide gel electrophoresis is
required for detection.

III.Shake bacteria and IPTG induction [Sirui Dong][Qiyu Tan][Rui
Yao][Saiyu Luo]

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image187.png)

For the group waiting for IPTG induction,add 40μL IPTG(c=0.5M)into
each conical flask and shake at 37℃,180rpm for another 4h

Centrifuge the bacteria solution after IPTG induction and store the
precipitation at -80℃

IV.Preparation of LB medium [Rui Yao][Qiyu Tan]

Follow protocol:LB liquid medium

We prepare 1.1L LB medium in total

V.Shake bacteria:group of miR-142[Qiyu Tan][Rui Yao]

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image188.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image189.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image190.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image191.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image192.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image193.png)

2024.09.17 Tuesday

Outline:

I.Vigorous maxiprep, digestion and agarose gel electrophoresis:㉘-2
;㉛[Rui Yao][Yuxiao Qin]

II. IPTG induction:㉒,㉓,㉔,⑬+㉒,㉗[Qiyu Tan][Sirui Dong]

III.Ultrasonic disruption of bacteria and fluorescence
measurement:⑬+㉛,⑯+㉛,㉛[Qiyu Tan]

IV.Store bacteria:㉒,㉓,㉔,㉗,⑬+㉒,⑬+⑯+㉛,⑬+⑯+㉘[Qiyu Tan]

V.Agarose gel electrophoresis of RNA:⑦,⑮,control,positive
control[Huixin Liang][Yuetong Ge]

VI.RNA extraction in vitro:③,㉗,㉞ [Huixin Liang][Yuetong Ge]

VII.Subsequent part of WB and coomassie staining of the
gel:PC,④,㉟[Huixin Liang][Yuetong Ge]

VIII.Vigorous maxiprep:group of miR-142[Qiyu Tan]

IX.Shake bacteria[Qiyu Tan]

Detailed experiments:

I.Vigorous maxiprep, digestion and agarose gel electrophoresis:㉘-2
;㉛[Rui Yao][Yuxiao Qin]

Vigorous maxiprep

Follow protocol:Vigorous plasmid maxiprep

Results

blank concentration = 0.059 ng/μL,

plasmid concentrations are as follow:

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image194.png)

Digestion

Follow protocol:Restriction Digest of Plasmid DNA

We used enzyme HpaI with rCutSmartBuffer.

We digest them at 37℃ for 1h.(A portion of the plasmid was digested
at 37 degrees for one hour)

The following volumes were added in the mixture:

The system are as follows

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image195.png)

Agarose gel electrophoresis

Follow protocol:Agarose gel electrophoresis

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image196.png)

description Picture

Store the plasmid

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image197.png)

Digest at 37℃

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image198.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image199.png)

The sample of agarose gel electrophoresis

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image200.png)

II. IPTG induction:㉒,㉓,㉔,⑬+㉒,㉗,⑳[Qiyu Tan][Sirui Dong]

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image201.png)

Shake at 220rpm for 4h before adding IPTG

Shake for another 4h after adding IPTG

Centriduge the bacteria solution after IPTG induction and store the
precipitation at -80℃

III.Ultrasonic disruption of bacteria and fluorescence
measurement:⑬+㉛,⑯+㉛,㉛[Qiyu Tan]

Ultrasonic disruption of bacteria

Follow protocol:Ultrasonic disruption of bacteria

Results

Blank:Abs=0.002,c=0.043

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image202.png)

Fluorescence mesurement

Follow protocol:Multi-Mode Microplate Reader

Dilute all the protein samples to 1.8 μg/μL to measure fluorescence

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image203.png)

Figure

description Picture

Measure the fluorescence

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image204.png)

IV.Store bacteria:㉒,㉓,㉔,㉗,⑬+㉒,⑬+⑯+㉛,⑬+⑯+㉘[Qiyu Tan]

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image205.png)

V. Agarose gel electrophoresis of RNA:⑦,⑮,control,positive
control[Huixin Liang][Yuetong Ge]

Follow protocol:Agarose gel electrophoresis of RNA

Loading order is as follows:(unit:μL)

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image206.png)

Run at 110V for 40min

We don't have marker so we use cr-21-5p as a marker

Result

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image207.png)

Analysis

Both of the control have clear band,which means our operation is
correct

⑦ has no band,Probably because the plasmids used(Tiangen maxiprep)
are impure

⑮ has clear band ,which means the transcription in vitro is
successful.

VI.RNA extraction in vitro:③,㉗,㉞ [Huixin Liang][Yuetong Ge]

Follow protocol: EasyPure® RNA Kit

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image208.png)

Store the RNA at -80℃

VII.Subsequent part of WB and coomassie staining of the
gel:PC,④,㉟[Huixin Liang][Yuetong Ge]

Follow protocol:Western Blot

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image209.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image210.png)

VIII.Vigorous maxiprep:group of miR-142[Qiyu Tan]

Follow protocol:Vigorous Plasmid Maxiprep

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image211.png)

IX.Shake bacteria[Qiyu Tan]

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image212.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image213.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image214.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image215.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image216.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image217.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image218.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image219.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image220.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image221.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image222.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image223.png)

2024.09.18 Wednesday

Outline:

I.Agarose gel electrophoresis:⑦(TIANGEN) [Huixin Liang][Yuetong Ge]

II.RNA extraction in vitro -I DNA template preparation:⑦(original
tube); ⑭(original tube) [Huixin Liang][Yuetong Ge]

III.Agarose Gel Electrophoresis [Saiyu Luo]

IV.Ultrasonic disruption of bacteria and fluorescence measurement
[Sirui Dong]

V.IPTG induction [Sirui Dong]

VI.Preparation of LB medium [Sirui Dong]

VII.Digestion and Agarose gel electrophoresis:group of miR-142[Qiyu
Tan]

VIII.Vigorous maxiprep [Qiyu Tan]

Detailed Experiments

I.Agarose gel electrophoresis:⑦(TIAN GEN)

Follow the protocol:Agarose Gel Electrophoresis

We suspected that less RNA might be due to incomplete digestion, so
we run agarose gel to verify the effectiveness of the digestion
system

Digested plasmid samples were loaded into the gel as the table
below.

The concentration of the gel is 1%.

4μL of 6X loading buffer were added to all samples.

The loading order and system are shown in the following table

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image224.png)

Analysis of results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image225.png)

Template DNA is pure

The enzyme XbaI and the reaction system works.

II. RNA extraction in vitro

1. DNA template preparation:Single enzyme digestion(10μL system)

In order to obtain a specific length RNA, the plasmid must be
completely linearized.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image226.png)

Digest at 37℃ for 2h

III.Agarose Gel Electrophoresis

Use 1% agarose gel

Heating until melt completely. Add 20μL Gelstain.

Run at 135V for 40min.

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image227.png)

-0 means it's not digested by enzyme

-1 means it's digested by single enzyme

IV.Ultrasonic disruption of bacteria and fluorescence measurement
[Sirui Dong]

Ultrasonic disruption of bacteria

Follow protocol:Ultrasonic disruption of bacteria

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image228.png)

Fluorescence measurement

We measure the fluorescence of ㉗→BL21 with other protein in the
group of miR-210

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image229.png)

Results

V.IPTG induction :⑳[Sirui Dong]

2 tubes in total

7.5ml bacteria solution in each tube

Shake 4h before adding IPTG,and shake for another 4h after adding

Centrifuge the bacteria solution after IPTG induction and store the
precipitation at -80℃

VI.Preparation of LB medium [Sirui Dong]

Follow protocol:LB liquid medium

We prepare 3L LB in total

VII.Digestion and Agarose gel electrophoresis:group of miR-142[Qiyu
Tan]

Follow protocol:Restriction Digest of Plasmid DNA Agarose gel
electrophoresis

Digest at 37℃ for 75min

The system of digestion is as follows

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image230.png)

Use 1% agarose gel

Run at 135V for 40min

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image231.png)

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image232.png)

VIII.Vigorous maxiprep [Qiyu Tan]

Follow protocol:Vigorous Plasmid Maxiprep

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image233.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image234.png)

2024.09.19 Thursday

Outline:

I.Ultrasonic disruption of bacteria and fluorescence measurement
[Rui Yao][Yuxiao Qin][Qiyu Tan]

II.Cell-free system:⑦+㉞[Yuetong Ge][Huixin Liang]

III.Digestion and Agarose gel electrophoresis:group of double-arm
LIRA1 and group of double-arm LIRA2[Qiyu Tan]

Detailed Experiments

I.Ultrasonic disruption of bacteria and fluorescence measurement
[Rui Yao][Yuxiao Qin][Qiyu Tan]

Ultrasonic disruption of bacteria

Follow protocol:Ultrasonic disruption of bacteria

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image235.png)

Fluorescence measurement

We measure the fluorescence of ㉓→BL21 with other protein in the
group of miR-210

Results

Because we forgot to mix the solution well,so the protein
concentration in the solution was not uniform,and the result had a
large error.

II.Cell-free system:⑦+㉞[Yuetong Ge][Huixin Liang]

Build the system according to the following table(unit:μL)

Incubate at 37 ° C for 6h

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image236.png)

Use 25μL β-gal as positive control

Add 0.5μL CPRG(c=30ug/μL) to each system

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image237.png)

III.Digestion and Agarose gel electrophoresis:group of double-arm
LIRA1 and group of double-arm LIRA2[Qiyu Tan]

Follow protocol:Restriction Digest of Plasmid DNA Agarose gel
electrophoresis

Digest at 37℃ for 75min

The systems of digestion are as follows

LIRA1

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image238.png)

LIRA2

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image239.png)

Use 1% agarose gel

Run at 135V for 40min

LIRA1

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image240.png)

LIRA2

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image241.png)

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image242.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image243.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image244.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image245.png)

2024.09.20 Thursday

Outline:

I.IPTG induction:⑬+⑯+㉘,⑬+⑯+㉛ [Rui Yao][Yuxiao Qin][Qiyu Tan]

II.Fluorescence measurement:the group of miR-142[Qiyu Tan]

III.Digestion and Agarose gel electrophoresis:group of double-arm
LIRA5[Qiyu Tan]

IV.RNA extraction in vitro:⑭ [Huixin Liang][Yuetong Ge]

Detailed Experiments

I.IPTG induction:⑬+⑯+㉘,⑬+⑯+㉛ [Rui Yao][Yuxiao Qin][Qiyu Tan]

Add 40μL IPTG(c=0.5M) to each conical flask

II.Fluorescence measurement:the group of miR-142[Qiyu Tan]

We measure the fluorescence of ㉓→BL21 with other protein in the
group of miR-210

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image246.png)

III.Digestion and Agarose gel electrophoresis:group of double-arm
LIRA5[Qiyu Tan]

Follow protocol:Restriction Digest of Plasmid DNA Agarose gel
electrophoresis

Digest at 37℃ for 75min

The system of digestion is as follows

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image247.png)

Use 1% agarose gel

Run at 135V for 40min

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image248.png)

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image249.png)

IV.RNA extraction in vitro:⑭ [Huixin Liang][Yuetong Ge]

Follow protocol:RNA extraction in vitro

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image250.png)

2024.09.21 Friday

Outline:

I.Ultrasonic disruption of bacteria and fluorescence
measurement:⑬+⑯+㉘,⑬+⑯+㉛

II.Cell-free system:Double-arm LIRA5[Huixin Liang][Yuetong Ge]

Detailed Experiments

I.Ultrasonic disruption of bacteria and fluorescence
measurement:⑬+⑯+㉘,⑬+⑯+㉛

Follow protocol:Ultrasonic disruption of bacteria

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image251.png)

Fluorescence measurement

Dilute all protein samples to 1.8 μg/μL by PBS, and add 100μL to
each hole to measure EGFP fluorescence.

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image252.png)
![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image253.png)

II.Cell-free system:Double-arm LIRA5[Huixin Liang][Yuetong Ge]

We construct the cell-free system according to the table
below(unit:μL)

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image254.png)

Add 0.5μL CPRG(c=300μg/μL) to each system

Use 25ul β-gal+0.5μL 25xCPRG as a positive control

Results

![](https://static.igem.wiki/teams/5038/img-notebook/exp-adj-2/image255.png)

2024.01.17 Wednesday

The First iGEM Team Meeting

After the establishment of the 2024 CJUH-JLU-China team, the members
of the Model team (Haoyang Liu , Xinhao Liao , Qing Ren , Yiting
Zhao) met for the first time and participated in the first iGEM team
meeting. Everyone introduced their familiar model, programming
language, and algorithms. We listened to the individual project
ideas of the experiment group and got a general idea about the
project. Since the topic has not yet been decided, the Model team
decided to learn about the models and algorithms that may be used,
and to learn from the excellent modeling examples of iGEM in the
past and try to reproduce them.

![](https://static.igem.wiki/teams/5038/img-notebook/model/1.png)
![](https://static.igem.wiki/teams/5038/img-notebook/model/2.png)

2024.01.21 Sunday

1st model group meeting

Due to the winter holiday, the first model inside meeting was held
in the form of an online meeting, and everyone put forward their own
interests in the direction.

Yiting Zhao learned about the work of the Virginia team at iGEM last
year, and introduced the principles of the kit and the modeling
principles.

Haoyang Liu tried to learn from the NCBI and TCGA databases,
explaining the data sources, data trustworthiness and how to search
them.

Qing Ren learned how to draw images with programming language and
tried to reproduce images from previous iGEM projects.

Xinhao Liao learned how to build a web page.

![](https://static.igem.wiki/teams/5038/img-notebook/model/3.png)

2024.01.26 Friday

2nd model group meeting

In order to better cooperate with other groups and clarify the
responsibilities of our group, we contacted Tuo Wang, the leader of
the previous model, asked him to pass on some experience to us.

Tuo Wang explained to us the various problems encountered by the
Model group in the last competition, and explained the final
research results to us. We took advantage of this opportunity to get
a better understanding of the task of the Model group.

2024.01.31 Wednesday

3rd model group meeting

This is the third model inside meeting , and the four team members
had completed their tasks excellently, and in this group meeting,
they shared their learning and reproduction results in details.

Qing Ren learned from the AutoDock software and constructed a
protein model.

Yiting Zhao explained the application of diffusion model in kits and
drug delivery, and introduced the model idea of HUST in 2022.

Xinhao Liao and Haoyang Liu explained the extraction, processing and
differential analysis of miRNA data in the database.

![](https://static.igem.wiki/teams/5038/img-notebook/model/4.png)

2024.02.18 Sunday

Progress acknowledgment and collaboration suggestion between Model
and Exp group on Heartecho project

For the first time, the Model group reported the progress of the
work with our PI , and after listening to the reports, our PI Xin Hu
and Youzhong Wan affirmed the progress of our group. They suggested
that the Model group communicate with the exp group in the early
stage of the next week's work, and suggested that the Model group
first cooperate with the exp group Zhongyu Chen's Heartecho project.

2024.02.24 Saturday

4th model group meeting

In the 4th model inside meeting ,members continued to share the
progress of the week's work.

Yiting Zhao studied the technical details of the HUST model,
focusing on the diffusion model, and tried to write code to simulate
the diffusion process.

Qing Ren explained the code and parameter adjustment of the
convolutional neural network, and tried to establish a prediction
model for the data set.

Xinhao Liao disassembled and studied the structure of last year's
web page, and completed the protein modeling attempt of HDOCK
software with Qing Ren together .

Haoyang Liu tried to construct a miRNA gene regulatory network and
analyze gene enrichment and survival.

![](https://static.igem.wiki/teams/5038/img-notebook/model/5.png)
![](https://static.igem.wiki/teams/5038/img-notebook/model/6.png)

2024.03.03 Sunday

Delegate and clarify tasks

We participated in the first all-staff meeting of the new semester.
We listened to the report of the exp group in full, and then
explained the progress of the model group to the PI and the students
of the exp group.

At the meeting, the exp group proposed to design a LIRA structure
that could detect miRNA, so the members of the model group were
divided into two groups.

Group A : Haoyang Liu and Xinhao Liao, who continue to be
responsible for the screening work.

Group B: Yiting Zhao and Qing Ren, who will inquire about software
that can meet the requirements of the experimental group.

![](https://static.igem.wiki/teams/5038/img-notebook/model/7.png)

2024.03.17 Sunday

Find databases and learn RNA design software

In this meeting, the Model Group presented the results of the
previous week.

Group A: Haoyang Liu and Xinhao Liao cross-compared the datasets of
various related heart diseases (heart failure, myocardial
infarction, coronary heart disease, hypertension) in the GEO
database and conducted difference analysis, sorted according to the
Fold Change value, and selected the top 20 miRNAs for
cross-comparison with various tumor databases, but did not find
miRNAs with good performance.

Group B: Yiting Zhao and Qing Ren studied NUPACK software and RNA
Strucure software, and tried to reproduce the single-arm structure
of H01 and H05 in the literature.

![](https://static.igem.wiki/teams/5038/img-notebook/model/8.png)
![](https://static.igem.wiki/teams/5038/img-notebook/model/9.png)

2024.03.24 Sunday

Search the database and start designing

In this presentation, members continued to report on the progress of
the week's work.

Group A: Haoyang Liu and Xinhao Liao expanded the search scope of
the GEO database dataset and found GSE104015 and GSE136547. We
identified the hsa-miR-223-3p and hsa-miR-18a-5p in the two datasets
as the target miRNAs. Model group recommended these two target miRNA
to exp group at the end of the meeting after discussion with PI.

Group B: Yiting Zhao and Qing Ren continued to simulate the
secondary structure of LIRA and started to design the LIRA structure
starting codon and RBS details with Zhongyu Chen.

![](https://static.igem.wiki/teams/5038/img-notebook/model/10.png)
![](https://static.igem.wiki/teams/5038/img-notebook/model/11.png)

2024.03.31 Sunday

Identify the miRNAs and start designing the game

Students in exp group conducted a literature search for our results,
and shared results with us on the meeting. They decided to expand
the search scope and continue searching works. Model group began to
design single-arm structure using miR-223-3p and miR-18a-5p
provisionally during the time waiting for exp group's result to
confirm our conclusions.

Qing Ren, Yiting Zhao, and Haoyang Liu were responsible for
designing part. We start from exploring the structure of miRNA
themselves.

Xinhao Liao began to be responsible for docking with the game design
of the students in the wiki group.

![](https://static.igem.wiki/teams/5038/img-notebook/model/12.png)

![](https://static.igem.wiki/teams/5038/img-notebook/model/13.png)
![](https://static.igem.wiki/teams/5038/img-notebook/model/14.png)

2024.04.06 Saturday

Find problems and try to find solutions

In this meeting, model group tried to investigate the proper
position to insert complementary sequence of target miRNA into
single-arm structure, and proper parameters. However, we met some
difficulties, small changes in position can have a huge impact on
secondary structure. We turn to PI for help in the meeting. PI
suggested us to cooperate with exp to find a more stable structure.

![](https://static.igem.wiki/teams/5038/img-notebook/model/15.png)

2024.04.14 Sunday

Conduct single-arm open-loop simulation and preliminary presentation
of the web page

In this meeting, we reported our progress on design, and attempts in
open loop simulation.

Qing Ren, Haoyang Liu and Yiting Zhao successfully found a feasible
method to construct a stable single-arm LIRA. After that, we use it
to do the open-loop simulation and didn't get expected result. We
think the problem existed in base pairs after discussion with exp
group.

Xinhao Liao presented the simulated renderings of the web pages
constructed based on the wiki drawings.

![](https://static.igem.wiki/teams/5038/img-notebook/model/16.png)

2024.04.21 Sunday

Double-arm gate design and search for new miRNA screening methods

We organized our miRNA differential analysis results and conducted
literature review on finding the evidence to support the
corresponding miRNA effect on certain diseases.

Qing Ren attempted to further analyze the double-arm gate structure
by introducing the influence of different orders of adding miRNAs on
the hybridization results. Although we have constructed a relatively
stable RNA open-loop simulation system, there are still some
unstable factors (mismatched bases) occurring during its operation,
it was found that when the two miRNAs were not added simultaneously,
it would cause changes in the results, which require further
improvement and optimization.

Xinhao Liao, Haoyang Liu and Yiting Zhao discussed with our PI
Youzhong Wan and Xin Hu, and the Model group decided to reconsider
the rationality of selecting the two miRNA indicators and attempt to
collaborate with the exp group to find support from epidemiological
evidence.

![](https://static.igem.wiki/teams/5038/img-notebook/model/17.png)

2024.04.27 Saturday

Broaden the search scope of the database and reproduce the structure
in the literature

We expand our searching criteria on atherosclerosis after we decided
to check whether we can find more outstanding miRNA in different
diseases.

Qing Ren, Yiting Zhao, and Haoyang Liu attempted to further modify
the double-arm gate structure, but have not yet found an effective
method. As a result, we re-examined the structures provided in the
references used for the LIRA design. Meanwhile, due to the lack of
epidemiological support for the selected miRNAs.

Haoyang Liu and Xinhao Liao attempted to broaden the search scope
for heart diseases and screen the datasets.

![](https://static.igem.wiki/teams/5038/img-notebook/model/18.png)

2024.05.05 Sunday

Illustrate the screening process with a flowchart and design a new
single-arm LIRA

We continue on our work on the identification of miRNA and organized
our process as flowcharts to demonstrate our workflow.

Haoyang Liu and Xinhao Liao attempted to organize the miRNA
screening process by drawing a flowchart. They also conducted
screenings on the GEO and TCGA databases, but have not yet obtained
the ideal target miRNAs.

Qing Ren and Yiting Zhao continued on their work on designing the
new single and double arm LIRA and attempt to build functional LIRA
by verifying the results after hybridizing the LIRA and new miRNA in
NUPACK Software.

![](https://static.igem.wiki/teams/5038/img-notebook/model/19.png)

2024.05.12 Sunday

Identify filtering metrics and try to design and learn machine
learning algorithms

We continued our working progress and made progress on the
identification of target miRNA.

Haoyang Liu and Xinhao Liao further expanded their search for heart
disease conditions to include myocardial infarction, and
comprehensively considered the FC values and the rankings of miRNAs
in heart diseases and tumors.

Yiting Zhao prepared for the subsequent analysis of the mathematical
model and began to learn machine learning algorithms start from
clustering.

Qing Ren started to design the double-arm LIRA to detect miR-192-5p
and miR-165-5p.

![](https://static.igem.wiki/teams/5038/img-notebook/model/20.png)

2024.05.19 Sunday

Determine the final miRNAs

We re-reviewing the LIRA research literature.

Xinhao Liao and Haoyang Liu concluded that miR-210-3p and miR-142-3p
are more suitable miRNAs. They presented the results of their
literature search collaboration with the exp team at this week's
group meeting, and found epidemiological evidence supporting the
role of miR-210-3p in myocardial infarction and tumors.

Qing Ren and Yiting Zhao decided to start designing various
single-arm structure which can be

used to detect miR-210-3p and miR-142-3p.

![](https://static.igem.wiki/teams/5038/img-notebook/model/21.png)

2024.05.29 Wednesday

Learn from previous years' gold medal projects to find where they
are available to use

All Model group members read and analyzed model parts on Indonesia's
wiki in 2023 together. We report it to our PI and discussed
feasibility of borrowing ideas from them. Our PI introduced results
Exp group obtained till that day. Model group decided to build our
own modeling ideas based on requirements and results raised by exp
group.

![](https://static.igem.wiki/teams/5038/img-notebook/model/22.png)

2024.06.12 Wednesday

Organize the overall project plan of the model group

In this meeting , the four members of the Model team decided to
organize our whole Model work plan to facilitate our work. We sorted
out the progress we had made and planned for the subsequent work,
successfully outlining a comprehensive work schedule for the Model.

All the members brought up their ideas on the process of modeling
the whole process of designing and evaluating the LIRA, we divided
our whole work into parts, the identification of our target miRNAs,
the designing of LIRA, the optimization of LIRA and finally the
simulation of the LIRA application in real world.

![](https://static.igem.wiki/teams/5038/img-notebook/model/23.png)

2024.07.07 Sunday

Introduce the model group's proposal to everyone on the team

After the Model group finished our finals, we partipated in the
all-staff meeting reunion to share the progress of each group. We
shared our progress and our working plan with our exp group and
continued our work.

2024.07.16 Tuesday

Start dynamic modeling

The Model group communicated with exp group, we began our work on
the kinetic model.

Haoyang Liu and Qing Ren introduced the Model group's work
arrangements, and further attempt to analyze the process of
designing single-arm LIRA and construct functional LIRA using our
previous selected miRNA.

Yiting Zhao and Xinhao Liao searched codes about kinetic simulation
and the principles during forming ordinary differential equations.

![](https://static.igem.wiki/teams/5038/img-notebook/model/24.png)

2024.07.25 Thursday

Design 6 types of single-arm LIRA and start building the wiki page

After continuous attempts, we made some progress.

Qing Ren and Haoyang Liu successfully constructed three stem-loop
ratio single-arm LIRA structures for miR-210-3p and three for
miR-142-3p.

Yiting Zhao tried to realize the kinetic model by Team-Indonesia and
used the information searched last week to list equations from
reaction steps listed by Team Indonesia. We compared our equations
with them, and solved the ODE equations by using MATLAB. The result
is the same and proves that the codes we used are basically correct.
We presented our requirements to the experimental group, requiring
them to tease out the chemical reaction process in the experiment
and list the chemical reaction expressions.

Xinhao Liao began constructing the wiki page. He first
systematically organized the components of the process of writing
the wiki. He started to searching for the tools in writing wiki and
organized the codes corresponding to different function.

![](https://static.igem.wiki/teams/5038/img-notebook/model/25.png)

2024.07.31 Wednesday

Proposed the concept of stem-loop ratio and continued working with
kinetics and wiki page

After designing multiple single-arm LIRA sequences, we attempt to
analyze the trends in these LIRAs' free energy change. And further
adjust our kinetic model.

Haoyang Liu and Qing Ren designed 13 different stem-loop ratios
(constrained by the LIRA structure) for the single-arm structures of
210-3p and 142-3p and conducted fitting curve simulations, but the
results were not satisfactory.

Yiting Zhao received reaction process of single-arm LIRA given by
Qiyu Tan and Sirui Dong. We listed out ODEs and done kinetic
simulations of single-arm LIRA.

Xinhao Liao piled up his work and organized each code for different
function such as the coloring of tables, inserting videos, etc. By
sorting out these sections, he greatly saved our time to optimize
our wiki.

![](https://static.igem.wiki/teams/5038/img-notebook/model/26.png)

2024.08.15 Thursday

Increase the number of LIRA designs, continue kinetic simulation and
upload the description page

We continue to explore the unsolved problems of last week, mainly
exploring the problems that cannot be explained by the setting of
stem-loop ratio and the results of dynamic simulations.

Qing Ren tried to design 560 single-arm LIRAs, and finally designed
a total of 280 single-arm LIRA sequences corresponding to the ratio
of 10 LIRA to each stem-loop ratio according to the stem ring
design, and the cluster analysis of these LIRAs did not find an
obvious linear relationship. Therefore, we deduced that the effect
of stem ring on the structure of single-arm LIRA was not
significant, and handed over to the exp group of 6 single-arm
sequences to verify our conclusion.

Yiting Zhao used reaction process provided by Qiyu Tan and Sirui
Dong to do the kinetic simulation of double-arm LIRA.

Xinhao Liao continued to build the wiki web page, he communicated
with the experimental team and uploaded the description page of our
project.

Haoyang Liu sorted out our process in LIRA design and organized it
into a pipeline. He also tried to further analyze the structure of
LIRA, and began to think about the unsatisfactory fitting effect of
the multivariate regression of the Indonesia team.

![](https://static.igem.wiki/teams/5038/img-notebook/model/27.png)
![](https://static.igem.wiki/teams/5038/img-notebook/model/28.png)

2024.08.21 Wednesday

Successfully designed a dual-arm LIRA and searched for the
parameters of the reaction process and communicated the wiki style

In this meeting, we try to design double-arm LIRA based on the
results of clustering stem-loop.

Haoyang Liu successfully designed double-arm LIRA by 7/15 and 8/15
and demonstrate the hybridization process of LIRA and 2 target
miRNA.

Qing Ren organized the single-arm designing results and communicate
with the exp group with our designed sequences.

Yiting Zhao discussed with Qiyu Tan and Sirui Dong about the part
that does not conform to the experimental expectations. Most of the
information in the literature describes extracellular, so we used k
value of extracellular to instead the lacking k value of
intracellular, which may resulted in inaccuracy.

Xinhao Liao communicated with the wiki group about the styles we
would like to present on our wiki page. They discussed how to
coordinate the visual message delivery with our project design.

2024.08.26 Monday

Find LIRA optimization methods and start writing wiki

In this meeting, we propose indicators to measure the performance of
LIRA, and try to construct an evaluation method for LIRA.

Xinhao Liao continues to make changes to the wiki and communicate
with the wiki about the style design of our website and made some
primary versions to compare.

Haoyang Liu and Yiting Zhao are inspired by Team Indonesia and
proposed that whether the RBS and AUG are exposed after the
hybridization of LIRA and miRNA can by analogizing to the expression
of EGFP in wet experiment. We introduce a concept of "lock" and
"unlock" to measure the performance of LIRA. We thought about how
LIRA's indicators measure LIRA, and proposed the importance of
introducing random forest models after calculated each indicator.

Qing Ren sorted out the previous single-arm design and double-arm
design content to start writing this part of the wiki content.

![](https://static.igem.wiki/teams/5038/img-notebook/model/29.png)

2024.09.08 Sunday

Upload the wiki content of the HP and Education groups and try to
draw a 3D model diagram

In this meeting, we successfully built a random forest model to link
the indicators to evaluate the performance of LIRA and the function
of LIRA.

Haoyang Liu tried to add different miRNAs to the kinetic simulation
model, and obtained the data on the change of EGFP expression in the
LIRA system when different concentrations of miRNAs were added. Also
tried our 3D curve analysis and wrote a difference analysis wiki
page.

Qing Ren completed the design process of single-arm and double-arm
LIRA writing content in the wiki.

Yiting Zhao began to try to write wiki contents for the LIRA
performance evaluation parts of the indicator with double-arm LIRA.

Xinhao Liao continued to build our websites, helping us to build the
initial website of Education and HP and brought up some suggestion
on the selection of materials and repaired some images during
interviews with less clarity.

2024.09.16 Monday

Start writing a notebook in the model group and upload the
Collaboration, Safety, and Protocol pages

Our model meeting is mainly to communicate about the writing of our
model page.

Haoyang Liu completed the writing of 3D wiki content and difference
analysis content.

Qing Ren organized the notebook of our model and assists in the
organization and modification of the content of the model's web
page.

Yiting Zhao completed the writing of the optimization and dynamic
simulation of double-arm.

Xinhao Liao continued to help our team with the construction of the
website, including completing the construction of the IHP website,
the collaboration website, the safety website, the protocol website,
and the start of the model website.

![](https://static.igem.wiki/teams/5038/img-notebook/model/30.png)

2024.09.18 Wednesday

The end of model

With our continuous efforts, our model work was successfully
completed. After the final proofreading of our wiki, our model work
was officially completed and we started to prepare our final
presentation.

Thank you for reading our notebook! We are heading for the best
Model!

https://2024.igem.wiki/cjuh-jlu-china/model

![](https://static.igem.wiki/teams/5038/img-notebook/model/31.png)

2023.01.17 Sunday

The first iGEM team meeting

This was the first time we held a meeting with all members in our
team, members in the Human Practice group and the Education group
analyzed the gold medal cases.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/1.jpg)

2024.01.25 Friday

1st brainstorm

The HP&Edu team shared our upcoming activity plans.

Luohan Ge shared her experiences of iGEM and provided guidance and
suggestions to the team members.

Jingying Qu proposed the idea of designing an original heart health
exercise routine.

Haiyue Yu proposed the idea of organizing a series of sports
activities, which was inspired by the spirit of the Paris 2024
Olympic Games.

Yeyao Guo proposed the idea of conducting educational presentations
tailored to students of different age groups.

Xinyi Shi proposed the idea of creating a children's picture book
and also prepared presentation slides for both elementary and high
school students.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/2.jpg)

2024.01.28 Sunday

Presentation at the First Middle School of Yitong Manchu Autonomous
County.

Luohan Ge delivered a lecture to the First Middle School of Yitong
Manchu Autonomous County.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/3.jpg)

2024.01.31 Wednesday

Presentation at Zibo No. 4 Middle School in Shandong Province.

Jingying Qu gave a lecture on the theme of synthetic biology and
iGEM competition at Zibo No. 4 Middle School in Shandong Province.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/4.jpg)

2024.02.02 Sunday

The sports activity plan.

Haiyue Yu finished the campus tennis teaching activity plan.

Xinyi Shi planned the "Draw the synthetic biology in our heart -
Frisbee DIY" activity.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/5.jpg)

2024.02.18-20 Tuesday-Thursday

1st WeChat press release

Jingying Qu wrote a press release on the lecture of Zibo No.4 Middle
School in Shandong Province.

Yeyao Guo typeset and Luohan Ge revised the WeChat official account
manuscript of Shandong Zibo No. 4 Middle School preach.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/6.jpg)

2024.02.21 Friday

Presentation at the Experimental School of Zichuan Economic
Development Zone

Jingying Qu chose the Experimental School of Zichuan Economic
Development Zone in Shandong Province to give an online lecture.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/7.jpg)

2024.02.22 Saturday

2nd brainstorm

Luohan Ge hosted the 2nd brain storm, and gave suggestions to the
group members.

Jingying Qu wrote the English interpretation version of the Cardiac
Health Exercises, Haiyue Yu modified.

Haiyue Yu proposed the idea of attending the 18thOriental Congress
of Cardiology, OCC2024.

Yeyao Guo proposed the idea of designing a "heart disease → tumor"
pipeline game.

Xinyi Shi proposed the idea of making the Synthetic Biology Manual.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/8.jpg)

2024.3.1 Friday

The activity plan

Haiyue Yu finished the attendance plan of the 18thOriental Congress
of Cardiology,OCC2024.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/9.jpg)

2024.3.2 Saturday

2nd WeChat press release

Luohan Ge finished the Wechat public number manuscript of the iGEM
theme lecture in the First Middle School of Yitong Manchu Autonomous
County, typesetting by Yeyao Guo.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/10.jpg)

2024.3.3 Sunday

Game design

Yeyao Guo completed the design and preliminary construction of the
"heart disease → tumor" pipeline game.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/11.jpg)

2024.3.4 Monday

Synthetic biology manual

Xinyi Shi wrote the text part of the synthetic biology manual, which
was modified by Luohan Ge and Zhongyu Chen.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/12.jpg)

2024.3.9 Saturday

3rd WeChat press release

Yeyao Guo completed the typesetting of the Wechat public account on
the theme of "Walking into iGEM" in Changchun No. 17 Middle School,
which was wrote by Yunze Li.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/13.jpg)

2024.3.9 Saturday

SYNO card game

Xinyi Shi adapted the UNO card game and combined it with synthetic
biology to form SYNO card game; Zhongyu Chen, Luohan Ge and Saiyu
Luo modified it.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/14.jpg)

2024.3.10 Sunday

4th WeChat press release

Yeyao Guo completed the typesetting of the WeChat official account
of the 2024 CJUH-JLU-China team recruitment activity.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/15.jpg)

2024.3.10 Sunday

Lecture at Shuxun Primary School in Nanguan District, Changchun

Yeyao Guo, Luohan Ge, Xinyi Shi, Haiyue Yu went to the Shuxun
Primary School in Nanguan District, Changchun, and gave a lecture on
the theme of "Into the Kingdom of Cells".

![](https://static.igem.wiki/teams/5038/img-notebook/hp/16.jpg)6

2024.3.13 Wednesday

1st Vlog

Haiyue Yu produced the team's first Vlog - Shuxun Primary School
lecture Vlog, which was modified by Luohan Ge.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/17.jpg)

2024.3.14 Thursday

The activity plan

Jingying Qu finished the writing the plan of the “Design your heart”
activity.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/18.jpg)

2024.3.15 Friday

The Synthetic Miracle Characters" theme blog

Yeyao Guo completed the writing of the Chinese manuscript of the
"Synthetic Miracle Characters" theme blog, and Luohan Ge, Xinyi Shi
modified it.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/19.jpg)

2024.3.15 Friday

The first draft of the picture book

Xinyi Shi wrote the first draft of the picture book "Little Noah who
doesn't love hygiene", which was revised by Luohan Ge and translated
by Haiyue Yu.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/20.jpg)

2024.3.17 Sunday

The recording of Cardiac Health Exercises

Jingying Qu, Yeyao Guo, Haiyue Yu and Luohan Ge recorded the full
version of Cardiac Health Exercises and the explanation video.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/21.jpg)

2024.3.17 Sunday

Feedback of education activities

Xinyi Shi wrote a team feedback form for educational activities, and
collected 866 feedback forms.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/22.jpg)

2024.3.17 Sunday

Introduced the production methods of bacterial painting

Haiyue Yu and Luohan Ge connected with professors from the School of
Plant Science of Jilin University,and the professor of microbiology
introduced the production methods of bacterial painting and some
painting skills to our team.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/23.jpg)

2024.3.19 Tuesday

Presentation at the International Department of Changchun Jilin
University

Haiyue Yu and Jingying Qu went to the International Department of
Changchun Jilin University Affiliated Experimental School, and gave
an all-English lecture.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/24.jpg)

2024.3.19 Tuesday

Presentation at the Affiliated Middle School of Jilin University

Yeyao Guo and Luohan Ge went to the Affiliated Middle School of
Jilin University, and gave a lecture on the theme of " Approaching
Synthetic Biology".

![](https://static.igem.wiki/teams/5038/img-notebook/hp/25.jpg)

2024.3.20 Wednesday

Presentation at the Changchun No. 5 Middle School

Haiyue Yu went to Changchun Tianjiabing Experimental Middle School
(Changchun No. 5 Middle School) to give a lecture on the theme of
synthetic biology.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/26.jpg)

2024.3.21 Thursday

5th WeChat press release

Yeyao Guo completed the writing of the press release and the
typesetting of the WeChat official account of the theme of "
Approaching Synthetic Biology" in the Middle School Affiliated to
Jilin University, Luohan Ge revised.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/27.jpg)

2024.3.21 Thursday

6th WeChat press release

Jingying Qu completed the Wechat public number manuscript of Zichuan
economic Development zone experimental school lecture, Yeyao Guo
completed the typesetting, Luohan Ge revised.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/28.jpg)

2024.3.23 Saturday

7th WeChat press release

Haiyue Yu wrote a press release from the lecture of International
Department of Changchun Jilin University Affiliated Experimental
School, Yeyao Guo typeset and Luohan Ge revised.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/29.jpg)

2024.3.27 Wednesday

Activity plan

Jingying Qu preliminary design the heart puzzle game.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/30.jpg)

2024.3.27 Wednesday

“Design your heart” activity

Jingying Qu, Yeyao Guo and Haiyue Yu carried out the activity
“Design your heart” at the Affiliated Middle School of Jilin
University (junior high school).

![](https://static.igem.wiki/teams/5038/img-notebook/hp/31.jpg)

2024.3.28 Thursday

The "heart disease → tumor" pipeline game

Yeyao Guo completed the production of the first draft of the "heart
disease → tumor" pipeline game display.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/32.jpg)

2024.3.30 Saturday

8th WeChat press release

Haiyue Yu wrote a press release on the theme of "CJUH-JLU-China's
date with high school students in synthetic biology", which was
revised by Luohan Ge, Yeyao Guo typeset.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/33.jpg)

2024.3.30 Saturday

9th WeChat press release

Jingying Qu wrote a press release on the activity of "Design your
heart", which was revised by Luohan Ge, Yeyao Guo typeset.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/34.jpg)

2024.3.31 Sunday

Campus tennis teaching activity

Haiyue Yu, Luohan Ge, Yeyao Guo, Xinyi Shi and Jingying Qu hosted
the Campus tennis teaching activity in Song Zhiping Gymnasium of
Jilin University.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/35.jpg)

2024.3.31 Sunday

Activity plan

Haiyue Yu wrote the Laboratory Open Day - Bacterial Painting Tour
activity plan.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/36.jpg)

2024.4.1 Monday

Activity feedback

Jingying Qu organized the feedback of "Design your heart" activity.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/37.jpg)

2024.4.2 Tuesday

SYNO card-game revise

Xinyi Shi revised the introduction of SYNO card-game.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/38.jpg)

2024.4.3 Wednesday

Activity feedback

Haiyue Yu sorted the feedback of Campus tennis teaching activity.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/39.jpg)

2024.4.4 Thursday

1st podcast

Yeyao Guo recorded the first episode of the podcast of "Synthetic
Miracle Characters".

![](https://static.igem.wiki/teams/5038/img-notebook/hp/40.jpg)

2024.4.5 Friday

The investigation questionnaire

Jingying Qu initially completed the investigation questionnaire.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/41.jpg)

2024.4.5 Friday

10th WeChat press release

Yeyao Guo wrote and typeset a press release from the lecture of
Shuxun Primary School in Nanguan District, Changchun and Luohan Ge
revised.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/42.jpg)

2024.4.6 Saturday

Activity idea

Xinyi Shi put forward the idea of 21 days of good work and rest.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/43.jpg)

2024.4.8 Monday

The video of Cardiac Health Exercises

Jingying Qu replaced the video cover of Cardiac Health Exercises,
which was drawn by Binghan Yang, and completed the video of Cardiac
Health Exercises.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/44.jpg)

2024.4.9 Tuesday

2nd podcast

Yeyao Guo recorded the second episode of the podcast of "Synthetic
Miracle Characters".

![](https://static.igem.wiki/teams/5038/img-notebook/hp/45.jpg)

2024.4.10 Wednesday

3rd podcast

Yeyao Guo recorded the third episode of the podcast of "Synthetic
Miracle Characters".

Interview with Prof. Songli Mei, a doctoral advisor in medical
ethics at the School of Public Health, Jilin University

Haiyue Yu contacted Professor Songli Mei from the School of Public
Health at Jilin University. Luohan Ge, Zhongyu Chen, and Yeyao Guo
interviewed Professor Mei about medical ethic issues.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/46.jpg)

2024.4.12 Friday

Interview with Prof. Songli Mei, a doctoral advisor in medical
ethics at the School of Public Health, Jilin University

Haiyue Yu contacted Professor Songli Mei from the School of Public
Health at Jilin University. Luohan Ge, Zhongyu Chen, and Yeyao Guo
interviewed Professor Mei about medical ethic issues.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/47.jpg)

2024.4.13 Saturday

Presentation at the Changchun Jiefang Road Primary School

Haiyue Yu and Yeyao Guo went to Changchun Jiefang Road Primary
School to teach basic biological knowledge to the students.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/48.jpg)

2024.4.13 Saturday

11th WeChat press release

Haiyue Yu completed the news draft for the preach at Changchun
Jiefang Road Primary School, Yeyao Guo completed the typesetting,
Luohan Ge revised.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/49.jpg)

2024.4.14 Sunday

The "Synthetic Journey, 'Fly' You Can't Miss" activity

Xinyi Shi, Luohan Ge, Haiyue Yu, Yeyao Guo, and Jingying Qu jointly
held the "Synthetic Journey, 'Fly' You Can't Miss" activity at Jilin
University. Yuxin Deng designed the frisbee sticker layout.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/50.jpg)

2024.4.14 Sunday

The activity plan

Haiyue Yu wrote the planning proposal for the theme music meeting
"Protect the Heart".

![](https://static.igem.wiki/teams/5038/img-notebook/hp/51.jpg)

2024.4.16 Tuesday

Presentation in the Evolution Community

Yeyao Guo and Luohan Ge gave a thematic preach on "Working Hand in
Hand with Residents to Protect the Heart" to community workers in
the Evolution Community.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/52.jpg)

2024.4.17 Wednesday

The vlog of the "Synthetic Journey, 'Fly' You Can't Miss" activity

Xinyi Shi edited the vlog of the "Synthetic Journey, 'Fly' You Can't
Miss" activity.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/53.jpg)

2024.4.18 Thursday

The news draft for the thematic preach "Working Hand in Hand with
Residents to Protect the Heart"

Yeyao Guo completed the news draft for the thematic preach "Working
Hand in Hand with Residents to Protect the Heart".

![](https://static.igem.wiki/teams/5038/img-notebook/hp/54.jpg)

2024.4.18 Thursday

Interview with Prof. Huan Sun from the Department of Cardiology at
the China-Japan Union Hospital of Jilin University

Zhongyu Chen and Yeyao Guo interviewed Dr. Huan Sun from the
department of Cardiology at the China-Japan Union Hospital of Jilin
University.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/55.jpg)

2024.4.19 Friday

The activity plan

Jingying Qu completed the planning proposal for the Heart Puzzle
Game.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/56.jpg)

2024.4.19 Friday

12th WeChat press release

Yeyao Guo typeset and Luohan Ge reviewed the WeChat public account
manuscript of the interview with Professor Songli Mei.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/57.jpg)

2024.4.20 Saturday

Interview with Dr. Linlin Liu

Zhongyu Chen, Jingying Qu, Haiyue Yu interviewed Dr. Linlin Liu,
Presedent of the China-Japan Union Hospital of Jilin University, and
director of the Oncology Department

![](https://static.igem.wiki/teams/5038/img-notebook/hp/58.jpg)

2024.4.21 Sunday

A heart health science activity

Xinyi Shi and Jingying Qu participated in a heart health science
activity at Jianyin·Qixiangyuan Changxing Elderly Care Center,
demonstrating and teaching cardiac health exercises.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/59.jpg)

2024.4.23 Tuesday

The QR code for the online survey questionnaire

Jingying Qu created the QR code for the online survey questionnaire.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/60.jpg)

2024.4.23 Tuesday

13th WeChat press release

Xinyi Shi completed the news draft for the "Synthetic Journey, 'Fly'
You Can't Miss" activity, Yeyao Guo completed the typesetting,
Luohan Ge revised.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/61.jpg)

2024.4.26 Friday

14th WeChat press release

Haiyue Yu wrote the Wechat official account manuscript of the Campus
tennis teaching activity, Yeyao Guo typeset and Luohan Ge reviewed.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/62.jpg)

2024.4.27 Saturday

The vlog for the "Design your heart" activity

Jingying Qu completed the final editing of the vlog for the "Design
your heart" activity.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/63.jpg)

2024.4.30 Tuesday

The activity plan

Jingying Qu completed the planning proposal for the heart health
interview.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/64.jpg)

2024.5.1 Wednesday

The "Laboratory Open Day" activity

Haiyue Yu, Yeyao Guo, Binghan Yang, Yuxin Deng, and Yixuan Xiao
completed the "Laboratory Open Day" activity.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/65.jpg)

2024.5.1 Wednesday

4th podcast

Yeyao Guo recorded the fourth episode of the "Synthetic Miracle
Character Chronicles" podcast.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/66.jpg)

2024.5.2 Thursday

5th podcast

Yeyao Guo recorded the fifth episode of the "Synthetic Miracle
Character Chronicles" podcast.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/67.jpg)

2024.5.2 Thursday

The heart health interview vlog

Jingying Qu edited the heart health interview vlog.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/68.jpg)

2024.5.3 Friday

6th podcast

Yeyao Guo recorded the sixth episode of the "Synthetic Miracle
Character Chronicles" podcast.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/69.jpg)

2024.5.3 Friday

The heart health interview news release

Jingying Qu completed the heart health interview news release,
edited by Luohan Ge.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/70.jpg)

2024.5.5 Sunday

15th WeChat press release

Xinyi Shi completed the news release for the nursing home event,
Yeyao Guo formatted, and Luohan Ge reviewed.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/71.jpg)

2024.5.9 Thursday

The activity plan

Yeyao Guo formatted, and Luohan Ge reviewed the planning document
for the heart health expo interview.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/72.jpg)

2024.5.10 Friday

The atherosclerosis brochure

Xinyi Shi completed the content for the atherosclerosis brochure.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/73.jpg)

2024.5.11 Saturday

The heart health expo interview

Yeyao Guo and Jingying Qu completed the heart health expo interview.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/74.jpg)

2024.5.11 Saturday

The heart disease survey questionnaire

Jingying Qu released the heart disease survey questionnaire.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/75.jpg)

2024.5.12 Sunday

The activity plan

Xinyi Shi planned to collaborate with the U-Tokyo iGEM team from
Japan.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/76.jpg)

2024.5.15 Wednesday

The news release for the heart health expo interview

Yeyao Guo wrote, and Luohan Ge reviewed the news release for the
heart health expo interview.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/77.jpg)

2024.5.16 Thursday

The video for the heart health expo interview

Yeyao Guo produced, and Luohan Ge reviewed the video for the heart
health expo interview.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/78.jpg)

2024.5.17 Friday

The "Laboratory Open Day" vlog

Haiyue Yu produced the "Laboratory Open Day" vlog.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/79.jpg)

2024.5.18 Saturday

The news release for the "Laboratory Open Day"

Haiyue Yu wrote the news release for the "Laboratory Open Day,"
edited by Luohan Ge.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/80.jpg)

2024.5.18 Saturday

The data analysis of the survey questionnaire

Jingying Qu completed the data analysis of the survey questionnaire.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/81.jpg)

2024.5.20 Monday

Collaboration with U-Tokyo

U-Tokyo responded for the first time, confirming their intention to
collaborate, agreeing to help translate children's picture books,
and expressing a desire to communicate about the project.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/82.jpg)

2024.5.22 Wednesday

Presentation at Changchun University for the Elderly

Yeyao Guo gave a presentation on "Prevention and Treatment of Heart
Disease" at Changchun University for the Elderly.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/83.jpg)

2024.5.23 Thursday

The design of the heart puzzle

Jingying Qu improved the design of the heart puzzle.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/84.jpg)

2024.5.24 Friday

The news release for the survey questionnaire

Jingying Qu completed the news release for the survey questionnaire.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/85.jpg)

2024.5.24 Friday

The news release for the lecture at the University for the Elderly.

Yeyao Guo wrote, and Luohan Ge reviewed the news release for the
lecture at the University for the Elderly.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/86.jpg)

2024.5.27 Monday

Collaboration with U-Tokyo

The team responded to questions from U-Tokyo. The response content
was provided by Saiyu Luo, reviewed by Zhongyu Chen, and translated
by Ziming Zhong.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/87.jpg)

2024.5.31 Friday

A collaboration meeting with the UTokyo team.

Xinyi Shi, Zhongyu Chen, Ziming Zhong, Saiyu Luo, and Haiyue Yu held
a collaboration meeting with the UTokyo team.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/88.jpg)

2024.6.3 Monday

The online oncology cardiology conference

Jingying Qu attended the online oncology cardiology conference.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/89.jpg)

2024.6.19 Wednesday

Interview with Prof. Yunsheng Dong from the School of Philosophy and
Social Sciences at Jilin University

Haiyue Yu, Yeyao Guo, and Zhongyu Chen interviewed Professor
Yunsheng Dong, Director of the Department of Sociology at the School
of Philosophy and Social Sciences, Jilin University.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/90.jpg)

2024.6.29 Saturday

The 18th Oriental Cardiology Conference.

Luohan Ge, Zhongyu Chen, and Jingying Qu attended the 18th Oriental
Cardiology Conference.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/91.jpg)

2024.7.5 Friday

The activity-"A Day of an iGEMer"

Shixin Yi organized an activity-"A Day of an iGEMer" and
communicated with the group leaders.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/92.jpg)

2024.7.6 Saturday

The press release for the 18th Oriental Cardiology Conference

Jingying Qu completed the press release and summary for the 18th
Oriental Cardiology Conference, and the first meeting with the
mentor.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/93.jpg)

2024.7.10 Wednesday

The activity plan

Xinyi Shi planned the "21 days make a healthy heart" event,
tentatively scheduled for August.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/94.jpg)

2024.7.11 Thursday

The CCiC conference

Zhongyu Chen, Luohan Ge, He Sun, Haiyue Yu, Yeyao Guo, and Yuetong
Ge attended the CCiC conference.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/95.jpg)

2024.7.14 Sunday

The framework for the Education page

Xinyi Shi began designing the framework for the Education page.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/96.jpg)

2024.7.14 Sunday

The vlog for the CCiC conference

Haiyue Yu made a Vlog for the CCiC conference.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/97.jpg)

2024.7.15 Monday

A press release about attending the CCiC conference

Haiyue Yu wrote a press release about attending the CCiC conference,
which was revised by Luohan Ge and Saiyu Luo.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/98.jpg)

2024.7.16 Tuesday

Interview with Dr. Daoyuan Si

Zhongyu Chen, Luohan Ge, and Yeyao Guo interviewed cardiologist Dr.
Daoyuan Si, associate chief physician of Cardiology at the
China-Japan Union Hospital of Jilin University.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/99.jpg)

2024.7.18 Thursday

Interview with Dr. Beibei Du, the Associate Chief Physician of
Cardiology at the China-Japan Union Hospital of Jilin University

Luohan Ge and Zhongyu Chen interviewed cardiologist Dr. Beibei Du,
the Associate Chief Physician of Cardiology at the China-Japan Union
Hospital of Jilin University.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/100.jpg)

2024.7.18 Thursday

The content of "Ten Tips for Scientific Cancer Prevention"

Xinyi Shi started writing the content of "Ten Tips for Scientific
Cancer Prevention."

![](https://static.igem.wiki/teams/5038/img-notebook/hp/101.jpg)

2024.7.18 Thursday

Interview draft with Professor Yunsheng Dong

Haiyue Yu wrote the Chinese and English versions of the interview
with Professor Yunsheng Dong.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/102.jpg)

2024.7.19 Friday

Interview with Dr. Wenqi Zhang, Department of Cardiology at the
China-Japan Union Hospital of Jilin University

Zhongyu Chen, Haiyue Yu, and Jingying Qu interviewed Dr. Wenqi Zhang
from the department of Cardiology at the China-Japan Union Hospital
of Jilin University

![](https://static.igem.wiki/teams/5038/img-notebook/hp/103.jpg)

2024.7.19 Friday

16th WeChat press release

Yeyao Guo formatted and Luohan Ge reviewed the CCiC WeChat public
account.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/104.jpg)

2024.7.22 Monday

Interview with Dr. Weihua Zhang from the First Hospital of Jilin
University

Haiyue Yu, Zhongyu Chen, Luohan Ge, and Yeyao Guo interviewed Dr.
Weihua Zhang, and Yeyao Guo recorded and wrote the interview draft.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/105.jpg)

2024.7.22 Monday

Interview draft with Dr. Wenqi Zhang

Jingying Qu completed the interview draft with Dr. Wenqi Zhang.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/106.jpg)

2024.7.23 Tuesday

The synthetic biology ethics manual

Yeyao Guo completed the Chinese and English versions of the
synthetic biology ethics manual assigned by CJUH-JLU-China.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/107.jpg)

2024.7.24 Wednesday

The team song

Haiyue Yu created the team song.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/108.jpg)

2024.7.26 Friday

The second meeting with the mentor

The team held the second meeting with the mentor, and Haiyue Yu
summarized the meeting content.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/109.jpg)

2024.7.27 Saturday

Interview with Dr. Jian Wu

Jingying Qu, Luohan Ge and Zhongyu Chen attended the first North
Spring City Cardiology Conference and interviewed Professor Jian Wu
and Prof. Yunzeng Zou.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/110.jpg)

2024.7.27 Saturday

The "21 days make a healthy heart" event

Xinyi Shi began promoting the "21 days make a healthy heart" event.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/111.jpg)

2024.7.28 Sunday

The HP web-page framework

The HP group initially finalized the HP web-page framework with
Zhongyu Chen.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/112.jpg)

2024.7.29 Monday

The Vlog of the 18th Oriental Cardiology Conference

Jingying Qu completed the Vlog for the 18th Oriental Cardiology
Conference.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/113.jpg)

2024.08.01 Thursday

The meeting with Yiye-China

Luohan Ge, Zhongyu Chen, Jingying Qu, Xinhao Liao, Haoyang Liu
participated the meeting with Yiye-China online.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/114.jpg)

2024.8.1 Thursday

“21 days, let's make hearts healthier!”

Xinyi Shi initiated the "21 Days to a Healthier Heart!" activity.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/115.jpg)

2024.08.02 Friday

Interview record of Dr. Jian Wu.

Jingying Qu finished the interview record of Dr. Jian Wu.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/116.jpg)

2024.8.2 Friday

Invitation for collaboration with UTokyo

Xinyi Shi reached out to team UTokyo to explore collaboration on the
Ethics of Synthetic Biology.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/117.jpg)

08.04-09.10 Sunday

IHP wiki framework

Haiyue Yu, Zhongyu Chen, and Yeyao Guo brainstormed together and
wrote the initial text for the integrated human practices (IHP)
page. They spent a month refining the content, going through a total
of 16 drafts. With the help of many CJUH-JLU-China members, such as
Jingying Qu, Ziming Zhong, and Adila, they completed the written
portion of the IHP section.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/118.jpg)

2024.8.6 Tuesday

The completion of Ten Scientific and Practical Tips for Cancer
Prevention

Xinyi Shi consulted the literatures and completed the textual
content of “Ten Scientific and Practical Tips for Cancer
Prevention”.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/119.jpg)

2024.8.8 Thursday

Planning for iGEMers' Day

Xinyi Shi completed the planning of the iGEMers' Day and
communicated with other groups to determine the work content of each
group.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/120.jpg)

2024.08.13 Tuesday

The SWOT analysis

Jingying Qu finished writing the SWOT analysis.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/121.jpg)

2024.08.14-09.19 Wednesday

Collaboration wiki framework

Luohan Ge wrote the initial draft for the Collaboration wiki content
and designed the preliminary webpage framework. Haiyue Yu, Jingying
Qu, Haoyang Liu, and Xinyi Shi provided subsequent content additions
and modifications, while Adila refined the language of the document.
Over the course of more than a month, they revised the draft seven
times, ultimately completing the written section.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/122.jpg)

2024.08.15 Thursday

Interview draft with Dr. Huan Sun

Yeyao Guo completed the interview draft with Dr. Huan Sun.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/123.jpg)

2024.8.14 Wednesday

WeChat official account article for the 1st Northeast iGEM Meetup

Yeyao Guo formatted the WeChat official account article for the 1st
Northeast iGEM Meetup, which was written and reviewed by Luohan Ge.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/124.jpg)

2024.08.15 Thursday

Interview with lawyer Jiuhui Feng

Before interviewing the patients, Yeyao Guo and Jingying Qu
interviewed lawyer Jiuhui Feng.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/125.jpg)

2024.08.16 Friday

Interview with lawyer Kunyang Xue

To obtain professional advice on the product feasibility of
Heartecho from a business perspective, Yeyao Guo and Jingying Qu
conducted an online interview with Kunyang Xue.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/126.jpg)

2024.8.18 Sunday

The medals wiki design

Xinyi Shi designed the medals wiki.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/127.jpg)

2024.8.18 Sunday

WeChat official account article for My Little Octopus

Yeyao Guo formatted the WeChat official account article for the game
My Little Octopus, which was written by Ting Sun and reviewed by
Luohan Ge.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/128.jpg)

2024.08.18 Sunday

Interview record

Jingying Qu finished the interview record of Dr. Linlin Liu.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/129.jpg)

2024.8.20 Tuesday

Manuscript collection of Ethics of Synthetic Biology

We have gathered the Chinese and English manuscripts of the partner
teams' contributions for the Ethics of Synthetic Biology.
Additionally, the English manuscript from UTokyo was translated into
Chinese by CJUH-JLU-China. Yeyao Guo communicated with co-editors
for compiling the content, collaboratively establishing the writing
logic and content framework for the documents.Yeyao Guo has
completed the initial draft of the comprehensive Chinese and English
versions of Ethics of Synthetic Biology.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/130.jpg)

2024.8.22 Thursday

Interview with lawyer JunlingBai

To obtain professional advice on the product feasibility of
Heartecho from a legal perspective, Yeyao Guo and Haiyue Yu
conducted an online interview with lawyer Junling Bai.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/131.jpg)

2024.8.23 Friday

Invitation for Collaboration on the translation of Invisible
Bacteria with Evry-Paris-Saclay

Xinyi Shi contacted Envy-Paris-Saclay to collaborate on the
translation of Invisible Bacteria.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/132.jpg)

2024.8.25 Sunday

Interview with Hongyu Li

To obtain professional advice on the product feasibility of
Heartecho from a industrial perspective, Yeyao Guo conducted an
interview with Hongyu Li, the Secretary of Board of Changchun
High-Tech Industry (Group) Co.,Ltd.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/133.jpg)

2024.08.27 Tuesday

Interview with Prof. Jin

Jingying Qu, Haiyue Yu, Haoyang Liu interviewed professor Lina Jin.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/134.jpg)

2024.08.28 Wednesday

Interview record

Jingying Qu finished the interview record of Prof. Lina Jin.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/135.jpg)

2024.8.30 Friday

Initial web content for the Entrepreneur section

Yeyao Guo has drafted the initial web content for the Entrepreneur
section of iHP. A detailed analysis was conducted covering unmet
needs, stakeholders & potential customers, product feasibility, and
development plans.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/136.jpg)

2024.9.4 Wednesday

WeChat official account article for the 18th Oriental
Cardio-Oncology Conference

Yeyao Guo formatted the WeChat official account article for the 18th
Oriental Cardio-Oncology Conference, which was written by Jingying
Qu and reviewed by Luohan Ge.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/137.jpg)

2024.09.06 Saturday

Exploring the Micro World: What Are Bacteria?

Xinyi Shi and Haiyue Yu went to the Third Kindergarten Affiliated
with Jilin University and held an activity about bacteria.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/138.jpg)

2024.9.8 Sunday

The business plan of Heartecho

Guo Yeyao completed the business plan of Heartecho, which detailed
our analysis of the market, cost, risk and other important factors
of commercialization.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/139.jpg)

2024.9.8 Sunday

The joint presentation of Jilin University's five iGEM teams and
enterprises

Zhongyu Chen, Xinyi Shi, Qiyu Tan, Haiyue Yu and Jingying Qu
participated in the joint presentation of Jilin University's five
iGEM teams and enterprises.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/140.jpg)

2024.9.13 Sunday

Patient interview

Haiyue Yu, Zhongyu Chen, Yeyao Guo, and Qiyu Tan visited the
Cardiology Department at Jilin University's China-Japan Union
Hospital, where they coordinated with Dr. Ming Yu and Dr. Zhongfan
Zhang. After securing informed consent from the patients and their
families, they conducted interviews in a confidential setting.
During these discussions, they obtained invaluable insights and
suggestions from the patients.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/141.jpg)

2024.9.13 Sunday

Interview with representatives of Johnson & Johnson Medtech

To confirm the viability of our business plan, Zhongyu Chen, Xinyi
Shi, Haiyue Yu,Yeyao Guo, and Jingying Qu interviewed
representatives of Johnson & Johnson Medtech. Junming Yan, Senior
Clinical Specialist in Johnson & Johnson Medtech, evaluated
Heartecho's business plan and provides recommendations for
amendments.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/142.jpg)

2024.9.13 Friday

The presentation video PPT

Jingying Qu completed the presentation video PPT, Haiyue Yu revised
and wrote the content.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/143.jpg)

2024.09.14 Saturday

iGEMers' Day

Xinyi Shi, Yeyao Guo, Qiyu Tan, Haoyang Liu, Ziming Zhong, He Sun,
and Zhongyu Chen invited 15 freshmen and held the activity “iGEMers'
Day”.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/144.jpg)

2024.9.16 Monday

WeChat official account article for JLU iGEM Team Meetup

YeyaoGuo formatted the WeChat official account article for JLU iGEM
Team Meetup, which was written by Jingying Qu and reviewed by Luohan
Ge.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/145.jpg)

2024.9.19 Thursday

Ethics of Synthetic Biology

Yeyao Guo completed the typesetting and checking of Ethics of
Synthetic Biology. The official Chinese and English versions of this
document are completed.

![](https://static.igem.wiki/teams/5038/img-notebook/hp/146.jpg)

2024/1/7 Sunday

1st wiki group meeting

During the first group meeting, members of the Wiki team (He Sun,
Yixuan Xiao, Binghan Yang, Yuxin Deng Ting Sun ) mainly created the
initial logo design for the project and the logo design for the mini
game

Yixuan Xiao, Binghan Yanga and Yuxin Deng designed the first version
of the logo for the project.

Ting Sun designed the first version of the logo for the project

Project logo

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/1.png)

Game logo

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/2.png)

2024/1/17 Wednesday

The First iGEM Team Meeting

During the first group meeting after the establishment of the
cjuh-jlu china team, Wiki team members drew attribution and
collaboration pages, while the Wiki team leader assigned specific
tasks to each person.

Yixuan Xiao, Binghan Yanga ,Yuxin Deng and Ting Sun designed the
first version of the cover image for “Attributions”and
“Collaboration”.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/3.png)

2024/1/26 Friday

2nd wiki group meeting

During the second group meeting, the Wiki team made some new designs
and also made modifications to the previous design drawings.

Yixuan Xiao:

1. Designed more logos for our project

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/.4png)

2. Modified the logo of our project

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/5.png)

Yuxin Deng:

1.Edited our team flag

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/6.png)

Ting Sun:

1. Made a draft of a game for our project

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/7.png)

2024/2/8 Thursday

3rd wiki group meeting

During the third group meeting, members of the Wiki team ( Yixuan
Xiao, Binghan Yang ) mainly created the initial team uniform for the
project

Team uniform

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/8.png)

2024/2/16 Friday

4th wiki group meeting

During the third group meeting, members of the Wiki team ( Yixuan
Xiao, Binghan Yang ) mainly modified our team uniform for the
project

Team uniform

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/9.png)

2024/2/25 Sunday

5th wiki group meeting

During this period, the Wiki team made some new designs and made
modifications to the previous design drawings, while mainly editing
"attribution" and "collaboration".

He Sun:

1. Practiced drawing experimental diagrams

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/10.png)

Yixuan Xiao:

1. Designed the team flag during this period.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/11.png)

2. Edited the cover image for “Attributions”

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/12.png)

Binghan Yang:

1. Modified the “Collaboration” page and Attributions”page.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/13.png)

2. Drew sketches of our teachers

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/14.png)

Yuxin Deng:

1. Edited the cover image for “Attributions”

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/15.png)

2. Edited the cover image for “Collaboration”

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/16.png)

Ting Sun:

1. Modified the draft of our game

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/17.png)

2024/3/5 Tuesday

6th wiki group meeting

During the sixth group meeting, members of the Wiki team ( Yixuan
Xiao, Binghan Yang, Yuxin Deng ,Ting Sun ) mainly edited the logo
designed for the project , edited the cover image for
“Collaboration” and ”Attribution”, made new designs for “Home” and
“Notebook” ,designed posters for events related to our project,
modified our team logo and team flag and made a draft of two games
for our project.

Yixuan Xiao:

1. Changed the format of the logo

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/18.png)

2. Designed the first version of the cover image for “Home”and
“Notebook”.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/19.png)

Binghan Yang:

1. Made two drafts of the first version of the cover image for
“Home”

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/20.png)

2. Made two drafts of the first version of the cover image for
“Notebook”

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/21.png)

Yuxin Deng:

1. Modified team flag logo

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/22.png)

2. Edited our team flag

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/23.png)

3. Designed decorative graphics for frisbees

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/24.png)

4. Designed a poster for the frisbee event

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/25.png)25

5. Designed the first version of the cover image for “Education
Tree”

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/26.png)

Ting Sun:

1. Made a draft of a puzzle game for our project

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/27.png)

2. Made a draft of a digital pet game for our project

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/28.png)

2024/3/11 Monday

7th wiki group meeting

During the seventh group meeting the Wiki team made some new designs
for “Safety” and our frisbees event, they also made modifications to
the previous design drawings such as “Notebook”, ”Home” and the
games.

Yixuan Xiao:

1. Designed the first version of the cover image for “Safety”

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/29.png)

Binghan Yang:

1. Followed up on the home page and modified the notebook page.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/30.png)

Yuxin Deng:

1. Modified our team logo and team flag.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/31.png)

2. Edited decorative graphics of frisbees.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/32.png)

3. Edited the posters of the frisbee event and the tennis event

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/33.png)

Ting Sun:

1. Kept modifying the games

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/34.png)

2024/3/16 Saturday

8th wiki group meeting

During this time, the Wiki team made some new designs for a picture
book of our project and also made modifications to the previous
design drawings such as posters and pics for the digital pet game.

He Sun:

1. Produced drafts of some parts for the picture book of our project

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/35.png)

Binghan Yang:

1. Designed a poster for the frisbee event

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/36.png)

Yuxin Deng:

1. Designed sticker graphics for the frisbees and drew a poster for
the frisbee event

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/37.png)

Ting Sun:

1. Kept modifying the games

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/38.png)

2024/3/24 Sunday

9th wiki group meeting

At the ninth group meeting, the Wiki team made some new designs such
as stickers and a video cover ,they also made modifications to the
previous designs.

He Sun:

1. Designed stickers related to our project.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/39.png)

Yixuan Xiao:

1. Edited the logos of our project

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/40.png)

Binghan Yang:

1. Edited the poster for the frisbee event and tennis event.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/41.png)

2. Designed the video cover for a heart health exercise routine of
our project

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/42.png)

Yuxin Deng:

1. Edited our team flag

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/43.png)

2. Drew some pictures for the pipe game of our project

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/44.png)

3. Edited the picture of the frisbee

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/45.png)

4. Edited our team logo

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/46.png)

Ting Sun:

1. Edit the pictures of the two games

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/47.png)

2. Made a draft of the pictures of the card game

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/48.png)

2024/3/31 Tuesday

The Second iGEM Team Meeting

During the second iGEM team meeting, members of the Wiki group
mainly created stickers designed for the project and the graphics of
our unique card game. They also kept making modifications of
previous designs.

He Sun:

1. Designed stickers and sticky notes related to the project

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/49.png)

2. Arranged the layout for the first few pages of the picture book

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/50.png)

Yixuan Xiao:

1. Edited the logos of our project

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/51.png)

2. Edited the cover image of “Home”

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/52.png)

Binghan Yang:

1. Modified the poster of the tennis event

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/53.png)

Yuxin Deng:

1. Edited the pictures of the pipe game

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/54.png)

2. Designed icons for the card game

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/55.png)

Ting Sun:

1. Designed icons for the card game

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/56.png)

2. Kept drawing the pictures of the digital pet game

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/57.png)

2024/4/6 Saturday

10th wiki group meeting

During this period, Wiki team members mainly focused on illustration
design for related activities, game illustration design, and webpage
modifications

He Sun :

complete the card format for the card game and design frisbee
stickers and the design and layout of the picture book.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/58.png)

Yixuan Xiao:

modify the Collaboration page.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/59.png)

Ting Sun:

1. Draw the main interface title and buttons for the Little Octopus
game.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/60.png)

2. Draw octopus game accessories.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/61.png)

3. Draw an outdoor scene with a small octopus and indoor lighting
effects during the day.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/62.png)

4. Create a dynamic effect of a small octopus standing up.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/63.png)

Binghan Yang:

1.Modify the team uniform.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/64.png)

2.Revise the cover of the heart health exercise.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/65.png)

3.Card layout.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/66.png)

Yuxin Deng:

1. Modify the pattern of the "Sick Heart" and the "tumor" in the
Pipe Game.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/67.png)

2. Complete the "Device" and "System" patterns for the card game.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/68.png)

3. Complete the back of the card drawing.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/69.png)

2024/4/14 Sunday

11th wiki group meeting

During the eleventh group meeting, we created new design drawings
and team flags for the webpage, peripheral designs, and created
posters for the HP team's activities. We also made adjustments to
card related designs and created relevant design drawings for the
game.

Yixuan Xiao:

1. Draw the Safety page diagram.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/70.png)

He Sun:

1. Complete uniform customization and sticker typesetting.![](https://static.igem.wiki/teams/5038/img-notebook/wiki/71.png)

2. Design the Notebook.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/72.png)

Yuxin Deng:

1. Design the new team flag.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/73.png)

2. Modify the back of the card and draw the card box.![](https://static.igem.wiki/teams/5038/img-notebook/wiki/74.png)

3. Draw the pipeline game cover.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/75.png)

Binghan Yang:

1. Complete the heart exercise cover drawing.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/76.png)

2. Modify the Frisbee campaign poster.and draw a bacterial painting
poster.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/77.png)

Ting Sun:

1. Add to the tutorial of the small octopus game.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/78.png)

2. Key presses and physical strength strips are drawn.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/79.png)

3. Egg content

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/80.png)

4. Achievement ending page of effect.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/81.png)

5. Indoor light effect.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/82.png)

2024/4/21 Sunday

12th wiki group meeting

During this period, we mainly focused on overall layout and design
of the webpage, and continued to follow up and modify game and
webpage design drawings.

He Sun:

1. Conceived of the webpage section.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/8
          .png)

2. Modify the Notebook.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/84.png)

Yixuan Xiao:

1. Modify the safety and collaboration page.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/85.png)

2. Design the Tame page. Description page··,Description page, Human
Prctices page.and Medal page.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/86.png)

Binghan Yang:

1. Card typography.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/87.png)

2. Conceived of the web-page scheme.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/88.png)

Yuxin Deng:

1. Concise the web plan

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/89.png)

2. Draw the pipeline game cover.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/90.png)

3. Lesdiseased heart puzzle and Healthy heart jigsaw puzzle.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/91.png)

2024/4/27 Saturday

13th wiki group meeting

This week, we mainly designed mascots, followed up with rich
illustrations for web pages and games, and created posters and
cartoon character images

He Sun:

1. Help to modify the little octopus game tutorial.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/92.png)

2. Design the web page role and conceived of the webpage section.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/93.png)

Yixuan Xiao Yuxin Deng and Binghan Yang:

1. Design mascot.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/94.png)

Yixuan Xiao:

1. Modify the Safety page.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/95.png)

Yuxin Deng:

1. Modify the pipeline game cover.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/96.png)

2. Modify the healthy heart jigsaw puzzle and the diseased heart
jigsaw puzzle.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/97.png)

3. Design the navigation map.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/98.png)

Binghan Yang:

1. Modify the bacterial painting poster.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/99.png)

2. Modify the card typesetting.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/100.png)

3. Draw the loading diagram.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/101.png)

Ting Sun:

1. Modify the small octopus game map.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/102.png)

2. Perfect the small game tutorial.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/102.png)

3. Make a small octopus to eat with the dynamic effect.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/104.png)

2024/5/5 Sunday

14th wiki group meeting

The Wiki team added web pages and games, designed a questionnaire,
and formatted SYNO game cards.

He Sun:

1. was mainly responsible for the team photo and the Notebook
template.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/105.png)

Yixuan Xiao:

1. drew the heart disease questionnaire

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/106.png)

2. Designed a webpage directory

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/107.png)

Ting Sun:

1. Designed button illustrations for the Little Octopus game Binghan
Yang:

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/108.png)

2. Modified and completed the layout of the cards

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/109.png)

3. Designed character cards for the team page

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/110.png)

Yuxin Deng:

1. designed the navigation bar map of the web page.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/111.png)

2. Designed picture book illustrations

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/112.png)

2024/5/19 Sunday

15th wiki group meeting

This week, we updated the content of the picture book, the first
picture and content of the page, designed the autumn edition of the
autumn edition, and added background pictures of the media platform
account.

He Sun:

1. Colored the picture book of invisible Bacteria.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/113.png)

2. Drew the Manual of Synthetic Biology.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/114.png)

Yixuan Xiao:

1. Drew the first page of Model page,Medium page and Education page.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/115.png)

Binghan Yang:

1. Drew the avatars for the Team page members

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/116.png)

2. Designed the autumn team uniforms.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/117.png)

Yuxin Deng:

1. Updated the picture book Invisible Bacteria,

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/118.png)

2. Designed the blog cover and the background map of the TikTok
platform.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/119.png)

2024/5/26 Sunday

16th wiki group meeting

We updated the web page this week, with changes to previous designs,
adding new puzzle designs and podcast covers.

He Sun:

1. Painted the new line draft of the picture book "No Bacteria".

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/120.png)

2. Revised the layout of the synthetic biology manual.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/121.png)

Yixuan Xiao:

1. Drew the Engineering page and notebook page.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/122.png)

Binghan Yang:

1. Updated the profile pictures of the team member

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/123.png)

Yuxin Deng:

1. Modify the heart illustration puzzle

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/124.png)

2. Designed podcast cover illustrations

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/125.png)

2024/6/23 Sunday

During the final-exams month, we still finished some of the art work

Binghan Yang

1. Modify the fall uniform.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/126.png)

Yuxin Deng:

1. Designed what the card box would look like

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/127.png)

2. Perfected a podcast cover

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/128.png)

Xixuan Xiao:

1. Designed the CCiC promotion poster

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/129.png)

2024/7/14 Sunday

Continue updating web design, avatar design, picture book
illustrations, and game design progress.

During the July holiday, we continued to catch up with the progress
and made new designs in picture books, web pages, and games

He Sun:

1. Updated the drawing of the picture book "The Invisible
Bacterium".

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/130.png)

2. Make a web page list.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/131.png)

Yixuan Xiao:

1. Modified the color scheme for attributes, safety, and notebook
pages.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/132.png)

Binghan Yang:

1. Team page character card drawing.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/133.png)

Yuxin Deng:

1. Designed video illustrations.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/134.png)

Ting Sun:

1. Designed character roles for the Little Octopus game

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/135.png)

2024/7/28 Sunday

Continue with respective processes

In late July, we gradually completed the work of the first ten days
and designed new mascots and posters in the latter half of the
month.

Yixuan Xiao:

1. Designed a new mascot.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/136.png)

2. Design new forms of web pages.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/137.png)

Binghan Yang:

1. Created cartoon animations.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/138.png)

2. Created a check-in poster.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/139.png)

2024/8/3 Saturday

Continue to draw web pages and design small illustrations of web
content.

In August, we will continue to draw and modify the first image of
the webpage, while also starting to design small illustrations and
some activity illustrations within the webpage.

Sun He:

1. Customize peripherals.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/140.png)

2. Add multilingual versions of Uygur and Korean to the picture book
Invisible Bacteria.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/141.png)

3. Designed and drawn Education page Wider and Deeper images, as
well as PDCA loop images and cognitive development theory images.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/142.png)

4. Designed and drew an education tree image for the Education
webpage.And drew balloons and content on the education tree.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/143.png)

Yixuan Xiao:

1. Continued updating the Attributions webpage and the Safety
webpage header image.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/144.png)

Binghan Yang:

1. Designed the typesetting of SYNO cards.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/145.png)

2024/8/12 Monday

Continue to design illustrations and homepage images

During this period, we will continue to work in an orderly manner
according to each person's schedule.

Sun He:

1. Created a title image for the education page.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/146.png)

2. Design and draw the characters of Ciose the loop and the dialog
box in the web page.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/147.png)

3. They summarized and drew the statistical results of the
questionnaire.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/148.png)

4. Took pictures of the team's surroundings.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/149.png)

Yixuan Xiao:

1. Updated the Education webpage and was responsible for the
homepage image of the Responsible for the world page.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/150.png)

Binghan Yang:

1. SYNO card instructions.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/151.png)

Yuxin Deng:

1. Draw the sixth issue of the blog cover.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/152.png)

2024/8/18 Sunday

Update event illustrations, webpage illustrations, and event
illustrations

This week, we advanced web and game design, and also designed a team
baseball hat.

Sun He :

1.A picture book was added.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/153.png)

2. We have designed the iGEMers Day seal.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/154.png)

Xiao Yixuan :

1.Design the Integrated Human Practices page.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/155.png)

Ting Sun :

1.Make small octopus games.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/156.png)

Binghan Yang:

1. Design the team baseball caps.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/157.png)

2.The card was typified.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/158.png)

3.Typoed the card manual.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/159.png)

2024/8/24 Saturday

Continue to design webpage content and activity illustrations

We are gradually producing website content and manuals, rearranging
them

Sun He :

1. Designed the web drop pdown.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/160.png)

Yixuan Xiao:

1.Design the Education page.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/161.png)

Binghan Yang:

1.The Synthetic Biology Manual was typographed.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/162.png)

Deng Yuxin and Yang Binghan jointly drew the small animals in the
puzzle game.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/163.png)

2024/9/7 Saturday

Preparation of manuals and business plans

During this period, we have created a large number of web page icons
and illustrations, while continuing to update game illustrations.

He Sun:

1. Design webpage icons and illustrations

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/164.png)

Yixuan Xiao:

1. Cover image of business plan involved

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/165.png)

Binghan Yang:

1. Revised the layout of the manual.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/166.png)

Ting Sun:

1. Completed the cover and ending images of the Little Octopus game

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/167.png)

2024/9/13 Friday

Create webpage illustrations and manual layout

This week, we continued to work on website icons, manual layout, and
game manuals

He Sun:

1. Continue designing webpage icons and illustrations

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/168.png)

Binghan Yang:

1. Draw illustrations。

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/169.png)

Ting Sun:

1. Typesetting Octopus Game Manual

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/170.png)

2024/9/15 Sunday

Drawing of webpage identifiers

This week, we will continue to work on the internal icons of the
webpage and the layout of some manuals。

He Sun:

1. Integrated Human Practices diagram has been created

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/171.png)

Binghan Yang:

1. Design loading background.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/172.png)

2024/9/21 Saturday

Manual drawing, manuscript writing, and page drawing

In late September, we almost completed the design of the first image
of the webpage and gradually improved the small and icons inside the
webpage. At the same time, the Little Octopus game is coming to an
end。

He Sun:

1. Drafted the surroundings of iGEMer's Day and Home page materials

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/173.png)

Yixuan Xiao:

1. Made the background map of official account

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/174.png)

Binghan Yang:

1. Created a contribution page

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/175.png)

Yuxin Deng:

1. Designed the cover and back of the ethics manual, and formatted
both Chinese and English versions

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/176.png)

Ting Sun:

1. Written the Octopus Protocol manual, game objectives, and three
Octopus animations.

![](https://static.igem.wiki/teams/5038/img-notebook/wiki/177.png)

2024.1.17 Wednesday

Translation and Refinement of Team Member Introductions

Following our first iGEM team meeting, where all members met for the
first time and introduced themselves, two Pre team members (Ziming
Zhong, Adila) gathered self-introductions and personal photos from
everyone. We handled the translation and proofreading, then compiled
everything for use on the Wiki Team Members page.

Adila:collected the self-introduction drafts and personal photos of
the team members, providing an initial translation and organization.

Ziming Zhong:revised and polished the self-introduction drafts and
organized them for future reference.

![](https://static.igem.wiki/teams/5038/img-notebook/pre-1/1.jpg)

2024.2.25 Sunday

Initial Brainstorming and Planning for the Promotion Video

During the winter holiday, we brainstormed initial ideas for the
promotion video, including the innovative idea of incorporating a
"Heart Disease Song," and discussed them with the team members.

Adila:came up with fresh ideas for the promotion video shoot,
organized them, and submitted the summary to Ziming Zhong.

Ziming Zhong:summarized and presented the video shoot ideas, then
discussed them with the team members and PI to plan the initial tone
and direction for the promotion video.

![](https://static.igem.wiki/teams/5038/img-notebook/pre-2/1.jpg)

2024.02.28 Wednesday

Following the HP team's online event on Tencent Meeting, themed
"Journey into Synthetic Biology," with 265 middle school students
from Zichuan Economic Development Experimental School, we translated
the event report from Chinese to English, polished the details, and
stored it for future use in our Wiki.

Ziming Zhong: Completed a thorough review of the Chinese draft and
introduced fresh ideas, shaping the main theme. Additionally,
conducted a second round of revisions, proofreading, and refining
the English version of the event report.

Adila: Designed the overall writing framework and completed the main
translation work, made revisions and adjustments to refine the
content.

![](https://static.igem.wiki/teams/5038/img-notebook/pre-1/3.jpg)

2024.03.01 Friday

Following the successful completion of the "iGEM: Exploring the
World of Synthetic Biology" event at Zibo No. 4 High School, we
translated the event report from Chinese to English, carefully
reviewed and refined the content, and archived it for future
reference in our Wiki.

Adila: Revised the Chinese version of the event report and
translated it into English.

Ziming Zhong: Conducted a second round of revisions, proofreading,
and refining of the English version of the event report.

![](https://static.igem.wiki/teams/5038/img-notebook/pre-1/4.jpg)

2024.03.13 Wednesday

Translating and Refining the Popular Science Brochure

After the HP team completed the Chinese version of our team's
Synthetic Biology Popular Science Brochure Synthetic biology:The
"Lego" of biology, we handled the editing and revision of the
English version.

Adila: Completed the revisions for the Chinese version of the
science brochure and translated it into English.

Ziming Zhong: Completed the second round of revisions and polishing
for the English translation.

![](https://static.igem.wiki/teams/5038/img-notebook/pre-1/5.jpg)

2024.03.28 Thursday

Following the HP team's educational event at Yitong No.1 Middle
School, with the theme "What is iGEM?", we translated the event
report from Chinese to English, polished the content, and saved it
for future reference in our Wiki.

Adila: Revised the Chinese version of the event report and
translated it into English.

Ziming Zhong: Conducted a second round of revisions, proofreading,
and refining of the English version of the event report.

![](https://static.igem.wiki/teams/5038/img-notebook/pre-1/6.jpg)

2024.04.01 Monday

Refining the Picture Book Invisible Bacteria

We revised and polished the English manuscript of our team's
original children's picture book Invisible Bacteria.

Adila: Completed the first round of editing and revisions on the
children's picture book manuscript.

Ziming Zhong: Completed the final revisions and proofread the
manuscript of the children's picture book , submitted it to the Wiki
team members.

![](https://static.igem.wiki/teams/5038/img-notebook/pre-1/7.jpg)

2024.04.04 Thursday

After successfully hosting the "Step into Synthetic Biology"
education event at the International Department of the Experimental
School Attached to Jilin University, we translated the event report
from Chinese to English, reviewed and refined the content, and
archived it for future reference in our Wiki.

Adila: Revised the Chinese version of the event report and
translated it into English.

Ziming Zhong: Conducted a second round of revisions, proofreading,
and refining of the English version of the event report.

![](https://static.igem.wiki/teams/5038/img-notebook/pre-1/8.jpg)

2024.04.16 Tuesday

After the HP team completed the educational event at Shuxun Primary
School, titled "Entering the Kingdom of Cells," we translated the
event report from Chinese to English, made necessary revisions, and
archived it for future use in our Wiki.

Adila: completed a thorough review of the Chinese draft and made
adjustments to the overall logical structure, and then handled the
main translation work.

Ziming Zhong: Conducted a second round of revisions, proofreading,
and refining of the English version of the event report.

![](https://static.igem.wiki/teams/5038/img-notebook/pre-1/9.jpg)

2024.04.22 Monday

Following the HP team's seminar at Jilin University Affiliated
Middle School, titled "Synthesizing the Future: Science
Popularization Seminar," we translated the event report from Chinese
to English, refined the content, and saved it for future reference
in our Wiki.

Adila: Revised the Chinese version of the event report and
translated it into English.

Ziming Zhong: Carried out a detailed second review of the English
report.

![](https://static.igem.wiki/teams/5038/img-notebook/pre-1/10.jpg)

2024.04.28 Sunday

After the HP team completed the "Design the Heart in Your Mind"
educational workshop at Jilin University Affiliated Middle School,
we translated the event report from Chinese to English, refined the
content, and archived it for future use in our Wiki.

Adila: Revised the Chinese version of the event report and completed
the main translation work.

Ziming Zhong: Led a meticulous second review of the English version,
incorporating thorough proofreading and refinement to polish the
report.

![](https://static.igem.wiki/teams/5038/img-notebook/pre-1/11.jpg)

2024.05.03 Friday

After the HP team held the education outreach at Changchun No. 17
High School, themed "Discovering the Romance of iGEM: A Symphony of
Science and Art," we translated the event report from Chinese to
English, refined the content, and archived it for future reference
in our Wiki.

Adila: Revised the Chinese version of the event report and
translated it into English.

Ziming Zhong: Conducted a second round of revisions, proofreading,
and refining of the English version of the event report.

![](https://static.igem.wiki/teams/5038/img-notebook/pre-1/12.jpg)

2024.05.14 Tuesday

Translation and Refinement of SYNO Game instructions

Once the HP team created our innovative synthetic biology promotion
game SYNO, we translated the design principles and rules into
English, refining and polishing the content to perfection.

Adila: Completed the revisions for the Chinese version of the game
instructions and translated it into English.

Ziming Zhong: Completed the second round of revisions and polishing
for the English translation.

![](https://static.igem.wiki/teams/5038/img-notebook/pre-1/13.jpg)

2024.06.13 Thursday

After the successful completion of the "Exploring Microorganisms"
educational event at Jiefang Road Primary School, we translated the
event report from Chinese to English, refined the content, and
archived it for future reference in our Wiki.

Adila: Revised the Chinese version of the event report and
translated it into English.

Ziming Zhong: Conducted a second round of revisions, proofreading,
and refining of the English version of the event report.

![](https://static.igem.wiki/teams/5038/img-notebook/pre-1/14.jpg)

2024.07.15 Monday

1st group meeting on EDU page Discussion

Following the completion of the majority of our education
activities, we held our first meeting with HP team members to
exchange ideas for the layout and content of the Education page.
Through this Meeting, we developed an initial framework with seven
key sections, shaping the foundation for the page's structure and
writing.

Ziming Zhong: Helped develop the overall structure for the Education
section, suggesting themes including idea workshops and
multi-sensory learning. He also ensured that the content was
coherently organized and stayed actively involved in managing the
writing process.

Adila: contributed ideas to help establish the framework for the
Education section, suggesting section titles such as "Edutainment."
She also prepared a plan for translating the manuscript after the
framework was finalized.

![](https://static.igem.wiki/teams/5038/img-notebook/pre-1/15.jpg)

2024.08.06 Tuesday

First Round of EDU Document Revision

After establishing the framework, we carried out the first round of
English translation and revisions for the Education section. This
included carefully refining the language to ensure clarity,
coherence, and ensure it was polished and engaging, setting the
stage for future content development and finalization.

Adila: Completed the revisions for the Chinese version of the
education documents, ensuring accuracy and consistency, then
translated the content into English, maintaining the core meaning
and intent throughout.

Ziming Zhong: Took charge of refining and polishing the English
translation, focusing on improving clarity, readability, and overall
coherence for the final version.

![](https://static.igem.wiki/teams/5038/img-notebook/pre-1/16.jpg)

2024.08.14 Wednesday

Second Round of EDU Document Revision

Building on the first round of translation and revisions, and
considering the substantial volume of content in the Education
section, we performed a comprehensive second review. Through
meticulous polishing, we ensured that the content was not only
engaging and coherent but also fully optimized and ready for
seamless integration into the Wiki page.

Adila: Focused on correcting details in the translated text,
adjusting terminology, ensuring consistency in tone, and proposing
improvements for certain sections to make the content more engaging.
She also provided specific feedback on areas where clarity could be
enhanced.

Ziming Zhong: Concentrated on improving the overall flow of the
document, reworking sentence structures for better readability and
ensuring a logical progression of ideas. He also refined the
transitions between sections to create a cohesive narrative for the
Wiki page.

2024.08.18 Sunday

English Adaptation of "My Little Octopus"

After successfully developing and launching the Chinese version of
our original game "My Little Octopus," we began work on the English
adaptation.This included not only translating the language but also
adjusting the tone and expressions to ensure the game feels natural
and engaging for a global players.

Adila: managed the translation and localization of the game's
tutorials, settings, key gameplay elements, and dialogues for
English players.

![](https://static.igem.wiki/teams/5038/img-notebook/pre-1/17.jpg)

2024.08.20 Tuesday

2nd group meeting on IHP page Discussion

In this meeting, we worked with the HP team members to plan how to
present the IHP section on our Wiki page.We reviewed the activities
we've completed so far, refined the narrative structure to ensure
clarity and flow, and reached a shared understanding on how best to
present our work.

Ziming Zhong:offered suggestions for the design of the IHP page
layout, refined the written content, and suggested improvements for
the project's application aspects.

![](https://static.igem.wiki/teams/5038/img-notebook/pre-1/18.jpg)

2024.08.23 Friday

Translation of the handbook "Ten Practical Tips for Cancer
Prevention"

After the HP team completed the Chinese version of the "Ten
Practical Tips for Cancer Prevention" public health guide, we
translated and proofread the content to produce the English version,
ensuring it was clear and well-suited for global audience.

Adila: translated and adapted the Chinese manuscript into English,
refining and proofreading the content to ensure clarity and accuracy
throughout.

![](https://static.igem.wiki/teams/5038/img-notebook/pre-1/19.jpg)

2024.08.26 Monday

Promotion Video Creation

After thorough discussions with all team members and our PI, and
refining the initial design for the promotion video, we established
a clear direction for the shoot. Collaborating with the Wiki and
Experimental teams, we successfully completed both the production
and editing, bringing the final video to completion.

Ziming Zhong: responsible for writing the script for the promotion
video and developing the overall filming plan. He worked closely
with the Wiki and Experimental teams on editing, and also took
charge of composing and recording the opening song for the video.

![](https://static.igem.wiki/teams/5038/img-notebook/pre-1/20.jpg)

2024.09.10 Tuesday

Revising the Collaboration Page

Adila: revised and restructured the layout of the collaboration
page, refined the English expression and improved the focus and
logical flow of the content.

![](https://static.igem.wiki/teams/5038/img-notebook/pre-1/21.jpg)

2024.09.22 Sunday

Preparation For the Presentation Video

After a thorough brainstorming session and extensive discussions
with all team members and our PI, we have finalized the shooting
plan for the presentation video and completed the script. With this,
all tasks of the Pre team have been successfully completed, and we
are now focusing on preparing for the final presentation. We are
excited to showcase our hard work at the upcoming Jamboree!

Ziming Zhong: revised and integrated the script for the presentation
video, developed the shooting plan, and managed the filming process.

![](https://static.igem.wiki/teams/5038/img-notebook/pre-1/22.jpg)

{% endblock %} {% block addJS %}
{% endblock %}

{% extends "layout.html" %} {% block addCSS %}
{% endblock %} {% block page\_content %}

Overview

Single-arm LIRA

Design

Build

Test

Learn

Double-arm LIRA

Design

Build

Test

Learn

Cell-free system

Design

Build

Test

Learn

Overview

We show 3 engineering cycles used in our project, i.e., single-arm
Loop-Initiated RNA Activator (LIRA), double-arm LIRA and cell-free
system. For each engineering cycle, it contains 4 stages——Design, Build,
Test and Learn. In the stage of Design, we make a blueprint to guide our
experiment. Following the design, we build our system in eligible
chassis. In the stage of Test, we verify our building by a variety of
experiments. Finally, we conclude what we have learned in the stage of
Learn.

Single-arm LIRA

Design

To develop an approach to screen cancer risk of post myocardial
infarction (MI) patients, we designed LIRA to target miR-210-3p and
miR-142-3p (H210 and H142), two cancer-promoting miRNA that are highly
expressed in post MI patients. As shown in the Figure 1, LIRA contains a
recognition region, a ribosome binding site (RBS) and a start codon
(AUG). Basically, the recognition region of LIRA is a reverse
complimentary sequence of its target RNA. Without interaction with its
target RNA, LIRA forms a stem-loop structure, and the RBS and AUG are
locked in the stem region, which cannot initiate the translation of the
downstream reporter gene. In the presence of the target RNA of LIRA, the
recognition region of LIRA could form base-paring interaction with its
target RNA, which disrupts the stem-loop structure of LIRA and release
the RBS and AUG for translation of the reporter gene.

![](https://static.igem.wiki/teams/5038/img-engineer/1.png)

**Figure 1**. Illustration of LIRA system.

To design LIRA that target miR-210-3p and miR-142-3p, we basically put a
sequence that is reverse complimentary to these miRNAs at the
recognition region of LIRA. As shown in the Figure 1, the recognition
region of LIRA contains a\* and b\*. The recognition region b\* is designed
in the stem structure of LIRA, and the recognition region a\* is designed
in the loop structure of LIRA. We hypothesized that the ratio of the
nucleotides of recognition region that are in the stem structure of LIRA
to the nucleotides of recognition region that are in the loop structure
of LIRA (stem-loop ratio) is critical for the function of LIRA, as it
determines how effective LIRA could interact with its target RNA. To
test our hypothesis, we designed LIRA that targets to miR-210-3p or
miR-142-3p with different stem-loop ratios. For miR-210-3p, which has 22
nucleotides, we designed LIRAs with stem-loop ratios of 2/20, 7/15 and
10/12, and for miR-142-3p, which has 23 nucleotides, we designed LIRAs
with stem-loop ratios of 3/20, 8/15 and 11/12 (Table 1). We would like
to test whether stem-loop ratio might influence the function of LIRA. In
addition, these 6 LIRAs have different free energies, indicating that
they might have different stabilities (Table 1). Therefore, we would
like to test whether free energies of LIRA alone might influence the
function of LIRA as well.

**Table 1**. 6 different stem-loop ratio LIRA data

| Stem-loop ratio | Single-arm sequence | Conformation of LIRA alone | Conformation of LIRA hybridized  with miR-210-3p |
| --- | --- | --- | --- |
| 2/20 | GUCUAGGAUGUGGGGUCUCUUAGCCGCCGGCUCAGCCGCUGUCACACGCACAGAGAGUGGGUGGAGAGGAGAUUAAAUAUGAUAGAC | ![](https://static.igem.wiki/teams/5038/img-model/table6/5.png) | ![](https://static.igem.wiki/teams/5038/img-model/table6/6.png) |
| 7/15 | CGGUGGAAUUGAACGUCUUCUGAUGGUCAGCCGCUGUCACACGCACAGGAGCUCCGGCGGCGGAAGAGGAGAUGCGCGAUGACACUG | ![](https://static.igem.wiki/teams/5038/img-model/table6/15.png) | ![](https://static.igem.wiki/teams/5038/img-model/table6/16.png) |
| 10/12 | CUGUCGGAUAUCUCAUCUUUUACUCAGCCGCUGUCACACGCACAGAUAAAAAGUCAGUCGCUGAAGAGGAGAUGGUAUAUGAGAUAG | ![](https://static.igem.wiki/teams/5038/img-model/table6/21.png) | ![](https://static.igem.wiki/teams/5038/img-model/table6/22.png) |

| Stem-loop ratio | Single-arm sequence | Conformation of LIRA alone | Conformation of LIRA hybridized  with miR-142-3p |
| --- | --- | --- | --- |
| 3/20 | GUGGCUAAUCUGACGUCUUCUGAGAUCGCGUCCAUAAAGUAGGAAACACUACACGGACACGGUGAGAGGAGAUGAAAGAUGGGAAAC | ![](https://static.igem.wiki/teams/5038/img-model/table7/7.png) | ![](https://static.igem.wiki/teams/5038/img-model/table7/8.png) |
| 8/15 | GAAGCAAGUACUAGAUCUUCUAAGAUCCAUAAAGUAGGAAACACUACACGAAGCGGGAAGGGUUAGAGGAGAUUGGGUAUGAGGUUC | ![](https://static.igem.wiki/teams/5038/img-model/table7/17.png) | ![](https://static.igem.wiki/teams/5038/img-model/table7/18.png) |
| 11/12 | GUGUUAAAUGUAAUGUUUUCUGUCCAUAAAGUAGGAAACACUACAUACGACGAAUACUAGGGGGAGAGGAGAUGGAACAUGAGAUAC | ![](https://static.igem.wiki/teams/5038/img-model/table7/23.png) | ![](https://static.igem.wiki/teams/5038/img-model/table7/24.png) |

Build

We cloned these 6 LIRA sequences into plasmid pCOLADuet-1 (Figure 2). To
enhanced expression of LIRA system, we chose T7 promoter as our
promoter. The expression of reporter gene would be increased by IPTG
induction so that we can control reaction time. In addition, an EGFP
reporter gene was cloned downstream of LIRA. Therefore, a functional
LIRA would initiate the expression of EGFP in the presence of its target
miRNA. For the expression of miRNA, we cloned miR-210-3p and miR-142-3p
into the plasmid pET-15b and pCDFDuet-1 (Figure 3). Plasmid pCOLADuet-1,
pET-15b and pCDFuet-1 carry different antibiotic marker genes for
selection, and they also have mutually compatible origin of replication.
Therefore, they can be transformed into bacterium simultaneously, and
desired colonies can be selected by using different combinations of
antibiotics. All the constructs were synthesized by GenScript.

![](https://static.igem.wiki/teams/5038/img-engineer/2-3.png)

**Figure 2**. Plasmid maps of 6 LIRA.

Map H142 3/20: pCOLADuet-H142\_3/20-linker-EGFP (With device
***PT7-LIRA\_H142\_3/20-linker-EGFP-T7TE***).

Map H142 8/15: pCOLADuet-H142\_8/15-linker-EGFP (With device
***PT7-LIRA\_H142\_8/15-linker-EGFP-T7TE***).

Map H142 11/12: pCOLADuet-H142\_11/12-linker-EGFP (With device
***PT7-LIRA\_H142\_11/12-linker-EGFP-T7TE***).

Map H210 2/20: pCOLADuet-H210\_2/20-linker-EGFP (With device
***PT7-LIRA\_H210\_2/20-linker-EGFP-T7TE***).

Map H210 7/15: pCOLADuet-H210\_7/15-linker-EGFP (With device
***PT7-LIRA\_H210\_7/15-linker-EGFP-T7TE***).

Map H210 10/12: pCOLADuet-H210\_10/12-linker-EGFP (With device
***PT7-LIRA\_H210\_10/12-linker-EGFP-T7TE***).

![](https://static.igem.wiki/teams/5038/img-engineer/3-3.png)

**Figure 3**. Plasmid maps of 4 input.

Map miR-142-3p(3337bp): pCDFDuet-LIRA \_input142 (With device
***PT7-LIRA\_input142-T7TE***).

Map miR-142-3p(5554bp): pET-15b-LIRA\_input142 (With device
***PT7-LIRA\_input142-T7TE***).

Map miR-210-3p(3336bp): pCDFDuet-LIRA\_input210 (With device
***PT7-LIRA\_input210-T7TE***).

Map miR-210-3p(5553bp): pET-15b-LIRA\_input210 (With device
***PT7-LIRA\_input210-T7TE***).

Test

Constructs with LIRA (H142 or H210) were transformed into competent
BL21-DE3 bacterium either alone or with constructs with miRNA (input142
or input210), and the transformed bacterium were plated on LB agar
plates with corresponding antibiotics to select successfully transformed
colonies (Figure 4).

![](https://static.igem.wiki/teams/5038/img-engineer/4.png)

**Figure 4**. Plate for single-arm LIRA.

Next, we picked up colonies from the plates and grew these colonies in
LB medium. Plasmid DNA were extracted from these colonies and digested
with endonuclease. The digested plasmid DNA were analyzed by Agarose Gel
Electrophoresis, which confirmed that plasmids from these colonies have
correct sizes (Figure 5-6).

![](https://static.igem.wiki/teams/5038/img-engineer/5.png)

**Figure 5**. Restriction endonuclease digestion of H142 and input142
plasmids after co-transformation. 1 % agarose gel electrophoresis.

Lane 1: pCOLADuet-H142\_3/20-linker-EGFP (4102 bp) digested with

HpaI.

indicated a 4102 bp single band.

Lane 2: pCOLADuet-H142\_8/15-linker-EGFP (4102 bp) digested with

HpaI.

indicated a 4102 bp single band.

Lane 3: pCOLADuet-H142\_11/12-linker-EGFP (4102 bp) digested with

HpaI.

indicated a 4102 bp single band.

Lane 4: Marker, 10000 DNA Marker.

Lane 5: pCOLADuet-H142\_3/20-linker-EGFP (4102 bp) and pET-15b-input142
(5554 bp) digested with

HpaI.

indicated a 4102 bp band and a 5554 bp band.

Lane 6: pCOLADuet-H142\_8/15-linker-EGFP (4102 bp) and pET-15b-input142
(5554 bp) digested with

HpaI.

indicated a 4102 bp band and a 5554 bp band.

Lane 7: pCOLADuet-H142\_11/12-linker-EGFP (4102 bp) and pET-15b-input142
(5554 bp) digested with

HpaI.

indicated a 4102 bp band and a 5554 bp band.

Lane 8: Marker, 10000 DNA Marker.

Lane 9: pCOLADuet-H142\_3/20-linker-EGFP (4102 bp) and pCDFDuet-input142
(3337 bp) digested with

HpaI.

indicated a 4102 bp band and a 3337 bp band.

Lane 10: pCOLADuet-H142\_8/15-linker-EGFP (4102 bp) and pCDFDuet-input142
(3337 bp) digested with

HpaI.

indicated a 4102 bp band and a 3337 bp band.

Lane 11: pCOLADuet-H142\_11/12-linker-EGFP (4102 bp) and
pCDFDuet-input142 (3337 bp) digested with

HpaI.

indicated a 4102 bp band and a 3337 bp band.

![](https://static.igem.wiki/teams/5038/img-engineer/6.png)

**Figure 6**. Single restriction endonuclease digestion of H210 and
Input210 by HpaI after co-transformation. 1 % agarose gel
electrophoresis.

Lane 1: pCOLADuet-H210\_2/20-linker-EGFP (4102 bp) digested with

HpaI.

indicated a 4102 bp single band.

Lane 2: pCOLADuet-H210\_7/15-linker-EGFP (4102 bp) digested with

HpaI.

indicated a 4102 bp single band.

Lane 3: pCOLADuet-H210\_10/12-linker-EGFP (4102 bp) digested with

HpaI.

indicated a 4102 bp single band.

Lane 4: Marker, 10000 DNA Marker.

Lane 5: pCOLADuet-H210\_2/20-linker-EGFP (4102 bp) and pCDFDuet-input210
(3336 bp) digested with

HpaI.

indicated a 4102 bp band and a 3336 bp band.

Lane 6: pCOLADuet-H210\_7/15-linker-EGFP (4102 bp) and pCDFDuet-input210
(3336 bp) digested with

HpaI.

indicated a 4102 bp band and a 3336 bp band.

Lane 7: pCOLADuet-H210\_10/12-linker-EGFP (4102 bp) and pCDFDuet-input210
(3336 bp) digested with

HpaI.

indicated a 4102 bp band and a 3336 bp band.

Lane 8: Marker, 10000 DNA Marker.

Lane 9: pCOLADuet-H210\_2/20-linker-EGFP (4102 bp) and pET-15b-input210
(5553 bp) digested with

HpaI.

indicated a 4102 bp band and a 5553 bp band.

Lane 10: pCOLADuet-H210\_7/15-linker-EGFP (4102 bp) and pET-15b-input210
(5553 bp) digested with

HpaI.

indicated a 4102 bp band and a 5553 bp band.

Lane 11: pCOLADuet-H210\_10/12-linker-EGFP (4102 bp) and pET-15b-input210
(5553 bp) digested with

HpaI.

indicated a 4102 bp band and a 5553 bp band.

IPTG-induced expression of EGFP protein were conducted in these
transformed clones by culturing in LB with 0.1mM IPTG for 4h. After
IPTG-induction, these bacteria were collected by centrifugation and
disrupted by ultrasonic. We measured the concentration of protein in
cell lysate and then diluted them into 1.8μg/μL. 100μL cell lysate was
added to 96-well plate and fluorescence from EGFP was measured by
Multi-mode Microplate Reader (Ex:488 Em:525).

H142 3/20 and H142 8/15 showed no difference with or without the
input142. H142 11/12 showed significantly increased fluorescence with
input142. Therefore, H142 11/12 performed much better than H142 3/20 and
H142 8/15 on targeting miR-142-3p (Figure 7).

![](https://static.igem.wiki/teams/5038/img-engineer/7.png)

**Figure 7**. Fluorescence comparison of BL21-DE3 with H142 related
plasmids by Multi-mode Microplate Reader with OD Ex:488 Em:525. \*\*\*
indicates *p* < 0.001.

While H210 7/15 and H210 10/12 showed no difference with or without
input210, H210 2/20 showed significantly increased fluorescence with
input210. Therefore, H210 2/20 performed much better than H210 7/15 and
H210 10/12 on targeting miR-210-3p (Figure 8).

![](https://static.igem.wiki/teams/5038/img-engineer/8.png)

**Figure 8**. Fluorescence comparison of BL21-DE3 with H210 related
plasmids by Multi-mode Microplate Reader with OD Ex:488 Em:525. \*\*\*
indicates *p* < 0.001.

Learn

From the results of our test, we can draw the following conclusions.

1. For LIRA to miR-142-3p, the LIRA with stem-loop ratio of 11/12, which
is the LIRA with the highest stem-loop ratio, performs the best. For
LIRA to miR-210-3p, the LIRA with stem-loop ratio of 2/20, which is the
LIRA with the lowest stem-loop ratio, performs the best. Therefore, it
seems that stem-loop ratio might not have big influence on the function
of LIRA to miRNA.

2. For LIRA to miR-142-3p, the LIRA with free energy of -41.1, which is
the LIRA with the highest free energy, performs the best. For LIRA to
miR-210-3p, the LIRA with free energy of -66.61, which is the LIRA with
the lowest free energy, performs the best. Therefore, it seems that free
energy of LIRA might not have big influence on the function of LIRA to
miRNA.

Double-arm LIRA

Design

To detect miR-210-3p and miR-142-3p simultaneously, we designed LIRAs
with a double-arm structure that can target two target RNA (Figure 9).
Double-arm LIRA has two recognition regions, and each recognition region
could form base pairing with a target RNA. Ideally, only when both
recognition region of the double-arm LIRA forms base pairing with its
target RNA, it can destabilize the stem-loop structure of LIRA to expose
RBS and AUG to initiate the expression of downstream reporter genes.

![](https://static.igem.wiki/teams/5038/img-engineer/9.png)

**Figure 9**. Illustration of Double-arm LIRA system.

We used sequences that were reverse complimentary to miR-210-3p and
miR-142-3p as recognition region, and utilized NUPACK to designed 100
double-arm LIRAs. We simulated the hybridization process of these LIRA
sequences with miR-142-3p and miR-210-3p, and excluded the LIRA
sequences that would not meet the expectation of our design. To optimize
LIRA design, we introduced multiple indicators to calculate the score of
expectation of LIRA, and finally chose 3 LIRA sequences for experiment
test (Please see our
[Model](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D)
section Optimization of double-arm LIRA for detailed information). While
double-arm LIRA 2 and ,double-arm LIRA 5[BBa\_K5038016] have highest score of
expectation, double-arm LIRA 1 have a medium score of expectation.

**Table 2**. Double-arm LIRA Sequences

| Number | Sequence | Conformation of LIRA alone | Conformation of LIRA hybridized with  miR-210-3p and miR-142-3p | Score of expectation |
| --- | --- | --- | --- | --- |
| 1 | AGCCUCAGCGGUCGACCCGCGGUCCAUAAAGUAGGAAACACUACAUUUGUGGGGAGCGGGGUAUAUCAUCCUCUCCUAGUCAGCCGCUGUCACACGCACAGCGGCUGGAGAGGAGAAUAUGAUAUACGGGGCCGAGGGGGCU | ![](https://static.igem.wiki/teams/5038/img-model/table9/1.png) | ![](https://static.igem.wiki/teams/5038/img-model/table9/2.png) | 1.47 |
| 2 | GUCCACGUUGAGCGUCCAGUCCAUAAAGUAGGAAACACUACAUUUGUGGAAAGGAUGGGUGCCUGUCCGAUCUCAGCUUCAGCCGCUGUCACACGCACAGCGGCUGAAGAGGAGAUGAUAUGGGCGCCGACAACGCAGAU | ![](https://static.igem.wiki/teams/5038/img-model/table9/4.png) | ![](https://static.igem.wiki/teams/5038/img-model/table9/3.png) | 1.71 |
| 5 | CGGUUCCGCUAAGGCAUCAUCCAUAAAGUAGGAAACACUACAUUUAUGGAACAUGCCCAUGUUUAUCCGUUCUCGACUUCAGCCGCUGUCACACGCACAGUGGCUGAAGAGGAGAAUAUAUGGAUAUGAUGGUGGCCCCG | ![](https://static.igem.wiki/teams/5038/img-model/table9/9.png) | ![](https://static.igem.wiki/teams/5038/img-model/table9/10.png) | 1.89 |

Build

As the cloning of single-arm LIRA described in the previous engineering
cycle, We cloned the double-arm LIRA sequences into plasmid pCOLADuet-1
(Figure 10). To enhanced expression of LIRA, we chose T7 promoter as the
promoter of LIRA. An EGFP reporter gene was cloned downstream of LIRA.
Therefore, a functional double-arm LIRA would initiate the expression of
EGFP in the presence of both miR-210-3p and miR-142-3p. All the
constructs were synthesized by GenScript.

![](https://static.igem.wiki/teams/5038/img-engineer/10.png)

**Figure 10**. Plasmid maps of double-arm LIRA.

Map Double-arm LIRA 1: pCOLA-LIRA\_ANDgate1-linker-EGFP (With
device ***PT7-LIRA\_ANDgate1-linker-EGFP-T7TE***).

Map Double-arm LIRA 2: pCOLA-LIRA\_ANDgate2-linker-EGFP (With
device ***PT7-LIRA\_ANDgate2-linker-EGFP-T7TE***).

Map ,double-arm LIRA 5[BBa\_K5038016]: pCOLA-LIRA\_ANDgate5-linker-EGFP (With
device ***PT7-LIRA\_ANDgate5-linker-EGFP-T7TE***).

Test

We transform constructs with double-arm LIRA and miRNA to *E.coil*
BL21-DE3 by heat shock and selected transformed bacteria colonies by
selection on plates with combined antibiotics (Figure 11-13).

![](https://static.igem.wiki/teams/5038/img-engineer/11.png)

**Figure 11**. Plate for pCOLA-LIRA\_ANDgate1-linker-EGFP
related transformation (BL21-DE3). From left to right. Plate1:
pCOLA-LIRA\_ANDgate1-linker-EGFP selected by Kan.

Plate2: pCOLA-LIRA\_ANDgate1-linker-EGFP+pET-15b-input142
selected by Kan and Amp.

Plate3: pCOLA-LIRA\_ANDgate1-linker-EGFP+pCDFDuet-input210
selected by Kan and STC.

Plate4:
pCOLA-LIRA\_ANDgate1-linker-EGFP+pET-15b-input142+pCDFDuet-input210
selected by Kan and Amp and STC.

![](https://static.igem.wiki/teams/5038/img-engineer/12.png)

**Figure 12**. Plate for pCOLA-LIRA\_ANDgate2-linker-EGFP
related transformation (BL21-DE3). From left to right. Plate1:
pCOLA-LIRA\_ANDgate2-linker-EGFP selected by Kan.

Plate2: pCOLA-LIRA\_ANDgate2-linker-EGFP+pET-15b-input142
selected by Kan and Amp.

Plate3: pCOLA-LIRA\_ANDgate2-linker-EGFP+pCDFDuet-input210
selected by Kan and STC.

Plate4:
pCOLA-LIRA\_ANDgate2-linker-EGFP+pET-15b-input142+pCDFDuet-input210
selected by Kan and Amp and STC.

![](https://static.igem.wiki/teams/5038/img-engineer/13.png)

**Figure 13**. Plate for pCOLA-LIRA\_ANDgate5-linker-EGFP
related transformation (BL21-DE3). From left to right. Plate1:
pCOLA-LIRA\_ANDgate5-linker-EGFP selected by Kan.

Plate2: pCOLA-LIRA\_ANDgate5-linker-EGFP+pET-15b-input142
selected by Kan and Amp.

Plate3: pCOLA-LIRA\_ANDgate5-linker-EGFP+pCDFDuet-input210
selected by Kan and STC.

Plate4:
pCOLA-LIRA\_ANDgate5-linker-EGFP+pET-15b-input142+pCDFDuet-input210
selected by Kan and Amp and STC.

To verify whether our transformation was successful, we purified plasmid
DNA from colonies by maxiprep and digested the plasmid DNA by
restriction endonuclease. The digested plasmid DNA was analyzed by
agarose gel electrophoresis, which confirmed that the transformed
colonies have the right size plasmid DNA(Figure 14-16).

![](https://static.igem.wiki/teams/5038/img-engineer/14.png)

**Figure 14**. Single restriction endonuclease digestion of
Double-arm LIRA 1 related plasmid DNA from colonies by HpaI. 1% agarose
gel electrophoresis.

Lane1: Marker, 10000 DNA marker.

Lane2: Maxiprep of pCOLA-LIRA\_ANDgate1-linker-EGFP plasmids in
BL21-DE3. pCOLA-LIRA\_ANDgate1-linker-EGFP has a single HpaI
cutting site

and

indicated a 4157 bp single band of
pCOLA-LIRA\_ANDgate1-linker-EGFP cut by HpaI.

Lane3: Maxiprep of
pCOLA-LIRA\_ANDgate1-linker-EGFP+pCDFDuet-input210
co-transformation plasmids in BL21-DE3.
pCOLA-LIRA\_ANDgate1-linker-EGFP and pCDFDuet-input210 each has
a single HpaI cutting site.

The

indicated a 4157 bp band of pCOLA-LIRA\_ANDgate1-linker-EGFP and
a 3336 bp band of pCDFDuet-input210 cut by HpaI.

Lane4: Maxiprep of
pCOLA-LIRA\_ANDgate1-linker-EGFP+pET-15b-input142
co-transformation plasmids in BL21-DE3.
pCOLA-LIRA\_ANDgate1-linker-EGFP and pET-15b-input142 each has a
single HpaI cutting site.

The

indicated a 4157 bp band of pCOLA-LIRA\_ANDgate1-linker-EGFP and
a 5554 bp band of pET-15b-input142 cut by HpaI.

Lane5: Maxiprep of pCOLA-LIRA\_ANDgate1-linker-EGFP
+pCDFDuet-input210+pET-15b-input142 co-transformation plasmids in
BL21-DE3. pCOLA-LIRA\_ANDgate1-linker-EGFP, pCDFDuet-input210
and pET-15b-input142 each has a single HpaI cutting site.

The

indicated a 4157 bp band of pCOLA-LIRA\_ANDgate1-linker-EGFP, a
3336 bp band of pCDFDuet-input210 and a 5554 bp band of pET-15b-input142
cut by HpaI.

![](https://static.igem.wiki/teams/5038/img-engineer/15.png)

**Figure 15**. Single restriction endonuclease digestion of
Double-arm LIRA 2 related plasmid DNA from colonies by HpaI. 1% agarose
gel electrophoresis.

Lane1: Marker, 10000 DNA marker.

Lane2: Maxiprep of pCOLA-LIRA\_ANDgate2-linker-EGFP plasmids in
BL21-DE3. pCOLA-LIRA\_ANDgate2-linker-EGFP has a single HpaI
cutting site

and

indicated a 4155 bp single band of
pCOLA-LIRA\_ANDgate2-linker-EGFP cut by HpaI.

Lane3: Maxiprep of
pCOLA-LIRA\_ANDgate2-linker-EGFP+pCDFDuet-input210
co-transformation plasmids in BL21-DE3.
pCOLA-LIRA\_ANDgate2-linker-EGFP and pCDFDuet-input210 each has
a single HpaI cutting site.

The

indicated a 4155 bp band of pCOLA-LIRA\_ANDgate2-linker-EGFP and
a 3336 bp band of pCDFDuet-input210 cut by HpaI.

Lane4: Maxiprep of
pCOLA-LIRA\_ANDgate2-linker-EGFP+pET-15b-input142
co-transformation plasmids in BL21-DE3.
pCOLA-LIRA\_ANDgate2-linker-EGFP and pET-15b-input142 each has a
single HpaI cutting site.

The

indicated a 4155 bp band of pCOLA-LIRA\_ANDgate2-linker-EGFP and
a 5554 bp band of pET-15b-input142 cut by HpaI.

Lane5: Maxiprep of pCOLA-LIRA\_ANDgate2-linker-EGFP
+pCDFDuet-input210+pET-15b-input142 co-transformation plasmids in
BL21-DE3. pCOLA-LIRA\_ANDgate2-linker-EGFP, pCDFDuet-input210
and pET-15b-input142 each has a single HpaI cutting site.

The

indicated a 4155bp band of pCOLA-LIRA\_ANDgate2-linker-EGFP, a
3336 bp band of pCDFDuet-input210 and a 5554 bp band of pET-15b-input142
cut by HpaI.

![](https://static.igem.wiki/teams/5038/img-engineer/16.png)

**Figure 16**. Single restriction endonuclease digestion of
,double-arm LIRA 5[BBa\_K5038016] related plasmid DNA from colonies by HpaI. 1% agarose
gel electrophoresis.

Lane1: Marker, 10000 DNA marker.

Lane2: Maxiprep of pCOLA-LIRA\_ANDgate5-linker-EGFP plasmids in
BL21-DE3. pCOLA-LIRA\_ANDgate5-linker-EGFP has a single HpaI
cutting site

and

indicated a 4155 bp single band of
pCOLA-LIRA\_ANDgate5-linker-EGFP cut by HpaI.

Lane3: Maxiprep of
pCOLA-LIRA\_ANDgate5-linker-EGFP+pET-15b-input142
co-transformation plasmids in BL21-DE3.
pCOLA-LIRA\_ANDgate5-linker-EGFP and pET-15b-input142 each has a
single HpaI cutting site.

The

indicated a 4155 bp band of pCOLA-LIRA\_ANDgate5-linker-EGFP and
a 5554 bp band of pET-15b-input142 cut by HpaI.

Lane4: Maxiprep of
pCOLA-LIRA\_ANDgate5-linker-EGFP+pCDFDuet-input210
co-transformation plasmids in BL21-DE3.
pCOLA-LIRA\_ANDgate5-linker-EGFP and pCDFDuet-input210 each has
a single HpaI cutting site.

The

indicated a 4155 bp band of pCOLA-LIRA\_ANDgate5-linker-EGFP and
a 3336 bp band of pCDFDuet-input210 cut by HpaI.

Lane5: Maxiprep of pCOLA-LIRA\_ANDgate5-linker-EGFP
+pCDFDuet-input210+pET-15b-input142 co-transformation plasmids in
BL21-DE3. pCOLA-LIRA\_ANDgate5-linker-EGFP, pCDFDuet-input210
and pET-15b-input142 each has a single HpaI cutting site.

The

indicated a 4155 bp band of pCOLA-LIRA\_ANDgate5-linker-EGFP, a
3336 bp band of pCDFDuet-input210 and a 5554 bp band of pET-15b-input142
cut by HpaI.

Next, all clones were conducted IPTG-induced expression of EGFP protein
by culturing the transformed clones in LB with 0.1mM IPTG for 4h. After
IPTG-induction, the transformed clones were disrupted by ultrasonic. We
measure the concentration of protein in cell lysate and then diluted
them into 1.8μg/μL. 100μL cell lysate is added to 96-well plate and
measured by Multi-mode Microplate Reader (Ex:488 Em:525). Double-arm
LIRA 1 showed no difference with or without input210 and input142
(Figure 17). Double-arm LIRA 2 showed increased fluorescence with
input142 alone, indicating that Double-arm LIRA 2 could be activated by
miR-142-3p alone (Figure 18). ,double-arm LIRA 5[BBa\_K5038016] showed significantly
increased fluorescence only in the case that both input142 and input210
were present, indicating that the activation of ,double-arm LIRA 5[BBa\_K5038016]
requires both miR-210-3p and miR-142-3p (Figure 19).

![](https://static.igem.wiki/teams/5038/img-engineer/17.png)

**Figure 17**. Fluorescence comparison of BL21-DE3 with Double-arm
LIRA 1 related plasmids by Multi-mode Microplate Reader with OD Ex:488
Em:525.

![](https://static.igem.wiki/teams/5038/img-engineer/18.png)

**Figure 18**. Fluorescence comparison of BL21-DE3 with Double-arm
LIRA 2 related plasmids by Multi-mode Microplate Reader with OD Ex:488
Em:525. \*\*\* indicates *p* < 0.001.

![](https://static.igem.wiki/teams/5038/img-engineer/19.png)

**Figure 19**. Fluorescence comparison of BL21-DE3 with Double-arm
LIRA 5[BBa\_K5038016] related plasmids by Multi-mode Microplate Reader with OD Ex:488
Em:525. \*\*\* indicates *p* < 0.001.

Learn

The, double-arm LIRA 5[BBa\_K5038016], which has the highest score of expectation,
performed best, which demonstrates that our optimization of double-arm
LIRA with indicators works.

Cell-free system

Design

In the future, we would like to apply our LIRA-based screening approach
to screen blood samples from post-myocardial infarction patients, so as
to identify those patients with high risk of cancer. Since it is
unlikely that we could transform miRNAs from blood samples into
bacteria, we need to adapt our LIRA-based screening approach into a
cell-free system. In addition, for a screening approach, it would be
much easier to detect changes of color than expression of EGFP, which
requires machine to measure fluorescence. Therefore, we set to make two
improvements of our LIRA-based screening approach. First, we change the
reporter gene from EGFP to LacZ, which could hydrolyze CPRG (yellow)
into CPR (purple). Second, we use a cell-free system to express the LacZ
cloned in our LIRA constructs.

Build

To set up the cell-free system for LIRA, we first tried H01 LIRA that we
have successfully used in our experiments at the beginning. We replaced
the reporter gene from EGFP to LacZ in H01 LIRA construct, and have it
synthesized by GenScript company (Figure 20). For the cell-free system,
we chose the PURExpress® In Vitro Protein Synthesis from New England
BioLabs. We used Input01 construct for the expression of input RNA and
engineered H01 LIRA construct for the expression of LacZ following the
protocol provided by the manufacture.

![](https://static.igem.wiki/teams/5038/img-engineer/20.png)

**Figure 20**. Plasmid maps of H01 LacZ and input 01.

Map H01 LacZ: pCOLADuet-H01-linker-lacZ (With device
***PT7-LIRA\_H01-linker-lacZ-T7TE***).

Map Input 01: pCDFDuet-input01 (With device
***PT7-LIRA\_input01-T7TE***).

Test

We first added Input01 plasmid to the In Vitro transcription system
provided by the T7 High Yield RNA Synthesis Kit (Yeasen) and incubated
it at 37℃ for 4h to get Input01 RNA. Then, we added the H01 LacZ plasmid
with or without purified Input01 RNA to the cell-free system provided by
the PURExpress® In Vitro Protein Synthesis and incubated it at 37℃ for
2h. To validate that H01 LIRA can work in the cell-free system, we added
CPRG. The results could be visualized or quantified by measuring OD562
with a multi-mode microplate reader. While the sample with H01 plasmid
alone could not change the color of CPRG, indicating that LacZ was not
expressed, the sample with both H01 plasmids and Input01 RNA changed the
color of CPRG, indicating that LacZ was expressed in this sample (Figure
21). This result demonstrated that input01 RNA activated H01 LIRA and
initiated the expression of LacZ.

![](https://static.igem.wiki/teams/5038/img-engineer/21-1.png)

**Figure 21**. OD562 measurement results. From left to
right. Tube 1: pCOLADuet-H01-linker-lacZ in cell-free system with CPRG.
Tube 2: pCOLADuet-H01-linker-lacZ and input01 RNA in cell-free system
with CPRG. Tube 3: β-gal with CPRG. \*\*\* indicates *p* < 0.001.

Learn

This engineering cycle validated our idea of combining LIRA with a
cell-free system, which paved the way of applying our engineered LIRA
that targets miR-142-3p and miR-210-3p into a cell-free system.

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Verification of established LIRA system

Amplification and examination of plasmid DNA

Transformation of LIRA and Input RNA plasmids into BL21-DE3

Examination of EGFP expression

Fluorescence Measurement

Identify target miRNA for cancer screening in CVD patients

Test of single-arm LIRA

Test of single-arm LIRA that targets miR-142-3p

Test of single-arm LIRA that targets miR-210-3p

Test of double-arm LIRA that target both miR-142-3p and miR-210-3p

Test of cell-free system

Verification of established LIRA system

In this stage, our goal was to verify the effectiveness of a LIRA system
reported in the literature and explore the best conditions and
experiment setting to test LIRA before developing our own LIRA system
for cancer screening [1]. We chose H01 and H05, two LIRAs
that have been reported with good performance to conduct our
verification experiments [1]. We first transformed plasmids
that carried LIRA or target RNA of LIRA (input RNA) into BL21-DE3, and
then used IPTG to induce the transcription of LIRA and input RNA, and
finally examined EGFP expression in the IPTG treated BL21-DE3 with
Western Blot analysis and fluorescence cytometry.

Amplification and examination of plasmid DNA

Plasmids carrying H01 and H05 LIRA were synthesized according to the
sequences provided in the literature [1] (Figure 1).

![](https://static.igem.wiki/teams/5038/img-results/1.png)

**Figure 1**. Maps of plasmid H01, H05, input01, input05.

Map H01: pET-15b-H01-linker-EGFP (With device
***PT7-LIRA\_H01-linker-EGFP-T7TE***).

Map input01: pCDFDuet-input01 (With device
***PT7-LIRA\_input01-T7TE***).

Map H05: pET-15b-H05-linker-EGFP (With device
***PT7-LIRA\_H05-linker-EGFP-T7TE***).

Map input05: pCDFDuet-input05 (With device
***PT7-LIRA\_input05-T7TE***).

These plasmids were transformed into DH5-α for amplification.
Transformed clones were grown in LB medium. Plasmid DNA from these
clones were extracted by maxiprep, and subjected to digestion with
endonucleases. The digested plasmid DNA was analyzed by agarose gel
electrophoresis, which demonstrated that the sizes of these plasmids
were correct (Figure 2-3).

![](https://static.igem.wiki/teams/5038/img-results/2-3.png)

**Figure 2**. Restriction endonuclease digestion of H01 and H05
plasmids after transformation. 1 % agarose gel electrophoresis.

Lane1: Marker, 10000 DNA marker.

Lane2 3 4: H01 digested with EcoRI. H01 has a single EcoRI cutting site

and

indicated a 6359bp single band of H01 cut by EcoRI.

Lane5 6 7: H05 digested with EcoRI. H05 has a single EcoRI cutting site

and

indicated a 6359bp single band of H05 cut by EcoRI.

![](https://static.igem.wiki/teams/5038/img-results/3-2.png)

**Figure 3**. Restriction endonuclease digestion of input01 and
input05 plasmids after transformation. 1 % agarose gel electrophoresis.

Lane1: Marker, 10000 DNA marker.

Lane2: input01 digested with XbaI. Input01 has a single XbaI cutting
site

and

indicated a 3369bp single band of input01 cut by XbaI.

Lane3: input05 digested with XbaI. Input05 has a single XbaI cutting
site

and

indicated a 3369bp single band of input05 cut by XbaI.

Transformation of LIRA and Input RNA plasmids into BL21-DE3

To generate clones that have both LIRA plasmids and their corresponding
input plasmids, H01+input01 & H05+input05 plasmids were
co-transformed into BL21-DE3, an engineered type of *E.coli* for
protein expression. Plasmids from transformed BL21-DE3 were extracted by
maxiprep, and digested by endonucleases. Agarose gel electrophoresis of
the digested plasmids demonstrated that H01+input01 & H05+input05
plasmids were co-transformed successfully (Figure 4-5).

![](https://static.igem.wiki/teams/5038/img-results/4-3.png)

**Figure 4**. Double restriction endonuclease digestion of H01 and
Input01 by EcoRI and XbaI. 1 % agarose gel electrophoresis.

Lane1: Marker, 10000 DNA marker.

Lane2: H01 digested with EcoRI. H01 has a single EcoRI cutting site

and

indicated a 6359bp single band of H01 cut by EcoRI.

Lane3: Input01 digested with XbaI. Input01 has a single XbaI cutting
site

and

indicated a 3369bp single band of input01 cut by XbaI.

Lane4: Maxiprep of H01+ Input01 co-transformation plasmids in BL21-DE3
digested with both EcoRI and XbaI. H01 has a single EcoRI cutting site

and

indicated a 6359bp single band of H01 cut by EcoRI. Input01 has a single
XbaI cutting site

and

indicated a 3369bp single band of input01 cut by XbaI.

![](https://static.igem.wiki/teams/5038/img-results/5-3.png)

**Figure 5**. Double restriction endonuclease digestion of H05 and
input05 by EcoRI and XbaI. 1 % agarose gel electrophoresis.

Lane1: Marker, 10000 DNA marker.

Lane2: H05 digested with EcoRI. H05 has a single EcoRI cutting site

and

indicated a 6359bp single band of H05 cut by EcoRI.

Lane3: Input05 digested with XbaI. Input05 has a single XbaI cutting
site

and

indicated a 3369bp single band of Input05 cut by XbaI.

Lane4: Maxiprep of H05+ Input05 co-transformation plasmids in BL21-DE3
digested with both EcoRI and XbaI. H05 has a single EcoRI cutting site

and

indicated a 6359bp single band of H05 cut by EcoRI. Input05 has a single
XbaI cutting site

and

indicated a 3369bp single band of Input05 cut by XbaI.

Examination of EGFP expression

To compare EGFP expression in BL21-DE3 clones with LIRA plasmid, H01 and
input01 or H05 with input05, all BL21-DE3 clones were conducted
IPTG-induction of EGFP protein by culturing the transformed BL21-DE3 in
LB with IPTG 0.1mM for 4h.

The transcription of H01 and H05 LIRA is regulated by T7 promotor, which
requires T7 RNA polymerase. The transcription of T7 RNA polymerase in
BL21-DE3 is regulated by lacUV5 promoter. The plasmid carrying H01 and
H05 LIRA (pCOLADuet-1) has a gene that encodes Lacl protein, which can
suppress the lacUV5 promotor. As a result, transformation of H01 and H05
plasmid into BL21-DE3 would suppress any transcription that requires T7
RNA polymerase. IPTG can combine with LacI protein, and release the
suppression of LacI protein on lacUV5 promoter, which activates the
transcription of T7 RNA polymerase. Therefore, we could use IPTG to
induce the transcription of T7 RNA polymerase in BL21-DE3 transformed
with H01 and H05 plasmid, and the induced T7 RNA polymerase could
activate the transcription of LIRA and the EGFP reporter gene downstream
of LIRA. After IPTG induction, the H01 and H05 transformed BL21-DE3
clones were treated by ultrasonic disruption, and cell lysate was
subjected to SDS-PAGE and Western Blot analysis.

Result of SDS-PAGE on H01 and H01+Input01 transformed BL21-DE3 clones
demonstrated that a strong expression of a 27kDa protein in the
IPTG-induced H01+input01 clones. Result of Western Blot demonstrated
that the expression of EGFP protein in H01+input01 transformed BL21-DE3
clone is much higher than that of H01 transformed BL21-DE3 clone with or
without IPTG induction, indicating that Input01 RNA successfully changed
the structure of H01 and exposed the RBS and start codon in H01 for
translation of downstream EGFP.

![](https://static.igem.wiki/teams/5038/img-results/6-2.png)

**Figure 6**. Examination of EGFP protein of H01 Gate RNA. 12 %
agarose gel SDS-PAGE image of

H01.

indicated protein bands of 27kDa EGFP expressed by H01 Gate. Lane1:
Marker. Lane2:H01 uninduced by IPTG. Lane3: H01 induced by 0.1mM IPTG.
Lane4: H01+Input01 uninduced by IPTG. Lane5: H01+Input01 induced by
0.1mM IPTG.

![](https://static.igem.wiki/teams/5038/img-results/7-2.png)

**Figure 7**. Examination of EGFP protein of H01 Gate RNA. 10 %
agarose gel Western Blot of EGFP. Lane1:H01 uninduced by IPTG. Lane2:
H01 induced by 0.1mM IPTG Lane3: H01+Input01 uninduced by IPTG. Lane4:
H01+Input01 induced by 0.1mM IPTG.

Similarly, result of SDS-PAGE on H05 and H05+Input05 transformed
BL21-DE3 clones demonstrated that an increased expression of a 27kDa
protein in the IPTG-induced H05+input05 clones. Result of Western Blot
demonstrated that the expression of EGFP protein in H05+input05
transformed BL21-DE3 clone is slightly higher than that of H05
transformed BL21-DE3 clone with or without IPTG induction, indicating
that Input05 RNA might slightly change the structure of H01 and exposed
the RBS and start codon in H01 for translation of downstream EGFP.

![](https://static.igem.wiki/teams/5038/img-results/8-2.png)

**Figure 8**. Examination of EGFP protein of H05 Gate RNA.12 %
agarose gel SDS-PAGE image of

H05.

indicated protein bands of 27kDa EGFP expressed by H05 Gate. Lane1:
Marker. Lane2:H05 uninduced by IPTG. Lane3: H05 induced by 0.1mM IPTG
Lane4: H05+Input05 uninduced by IPTG. Lane5: H05+Input05 induced by
0.1mM IPTG.

![](https://static.igem.wiki/teams/5038/img-results/9-2.png)

**Figure 9**. Examination of EGFP protein of H05 Gate RNA. 10 %
agarose gel Western Blot of EGFP. Lane1:H05 uninduced by IPTG. Lane2:
H05 induced by 0.1mM IPTG Lane3: H05+Input05 uninduced by IPTG. Lane4:
H05+Input05 induced by 0.1mM IPTG.

Together, while the expression of EGFP protein in H01+input01
transformed BL21-DE3 clone was much higher than that of H01 transformed
BL21-DE3 clone, the expression of EGFP protein in H05+input05
transformed BL21-DE3 clone was only slightly higher than that of H05
transformed BL21-DE3 clone. Such result demonstrated that the
effectiveness of H01 LIRA that targets input01 RNA is much higher than
that of H05 LIRA that targets input05 RNA.

Fluorescence Measurement

To quantify the LIRA-mediated EGFP expression, the fluorescence of
protein lysate from all BL21-DE3 clones were measured by Multi-mode
Microplate Reader. To compare the effectiveness of H01 and H05
quantitatively, we used the On/Off ratio. The On/Off ratio of LIRA is
defined as ON-state reporter gene expression, which is LIRA-mediated
downstream gene expression in the presence of the corresponding input
RNA, divided by the OFF-state reporter gene expression, which is
LIRA-mediated downstream gene expression in the absence of the
corresponding input RNA.

The On/Off ratio of H01 is 28.554, while the On/Off ratio of H05 is
1.345. Comparing the fluorescence measurement results of H01 and H05,
the On/Off ratio of H01 is much greater than H05, indicating that H01 is
a much more effective LIRA. Therefore, we decided to develop our own
LIRA based on the structure of H01.

![](https://static.igem.wiki/teams/5038/img-results/10.png)

**Figure 10**. Fluorescence comparison of BL21-DE3 with H01 plasmid
or H01+input01 plasmids by Multi-mode Microplate Reader with OD Ex: 488
Em: 525. \*\*\* indicates *p* < 0.001.

![](https://static.igem.wiki/teams/5038/img-results/11.png)

**Figure 11**. Fluorescence comparison of BL21-DE3 with H05 plasmid
or H05+input05 plasmids by Multi-mode Microplate Reader with OD Ex: 488
Em: 525. \*\* indicates *p* < 0.01.

![](https://static.igem.wiki/teams/5038/img-results/12.png)

**Figure 12**. On/Off comparison of H01 and H05 under IPTG induction.

Identify target miRNA for cancer screening in CVD patients

Using a variety of bioinformatic analysis, our model group identified
miR-142-3p and miR-210-3p as ideal biomarkers for cancer screening in
post-myocardial infarction patients. We did Wound healing and Transwell
assays to confirm that these two miRNAs could promote the migration and
invasion of cancer cells. For detailed information of this part of work,
please see our
[Model](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D)
section.

Test of single-arm LIRA

Test of single-arm LIRA that targets miR-142-3p

Our model group designed single-arm LIRAs that target miR-142-3p.
Recognition region is an essential part of LIRA, which is a reverse
complementary sequence of the target RNA of LIRA, and the stem-loop
ratio of LIRA refers the number of nucleotides of recognition region
that are in the stem structure of LIRA to the number of nucleotides of
recognition region that are in the loop structure of LIRA (Please see
our
[Model](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D)
section for detailed information). Since we believed that stem-loop
ratio was crucial for the effectiveness of LIRA, we chose 3 LIRAs with
different stem-loop ratios for evaluation. We cloned these LIRAs into
pCOLADuet-1, and named these constructs H142\_3/20, H142\_8/15 and
H142\_11/12 respectively. In addition, we cloned the sequence of
miR-142-3p into plasmid pCDFDuet-1 and pET-15b for the expression of
miR-142-3p, and named these constructs input142.

**Table 1**. Plasmid for H142.

| Number | Plasmid description | Device |
| --- | --- | --- |
| 1 | pCOLADuet-H142\_3/20-linker-EGFP | PT7-LIRA\_H142\_3/20-linker-EGFP-T7TE |
| 2 | pCOLADuet-H142\_8/15-linker-EGFP | PT7-LIRA\_H142\_8/15-linker-EGFP-T7TE |
| 3 | pCOLADuet-H142\_11/12-linker-EGFP | PT7-LIRA\_H142\_11/12-linker-EGFP-T7TE |
| 4 | pCDFDuet-input142 | PT7-LIRA\_input142-T7TE |
| 5 | pET-15b-input142 | PT7-LIRA\_input142-T7TE |

All plasmids listed in Table 1 were transformed into BL21-DE3 (Figure
13) and plasmids from successfully transformed clones were verified by
digestion with restriction endonuclease and agarose gel electrophoresis
(Please see our [Engineering](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D) section for detailed information).

![](https://static.igem.wiki/teams/5038/img-results/13-2.png)

**Figure 13**. Plate for H142 related transformation.

Transformed BL21-DE3 clones was treated with 0.1mM IPTG for 4h and then
disrupted by ultrasonic-sound. Cell lysate was collected by centrifuging
and diluted to 1.8 μg/μL. The EGFP fluorescence from the diluted cell
lysate was measured at a medium-high PMT gain. The results showed that
the clones transformed with H142\_11/12 and input142 have highest
expression of EGFP than other clones.

![](https://static.igem.wiki/teams/5038/img-results/14.png)

**Figure 14**. Fluorescence comparison of BL21-DE3 with H142 related
plasmids by Multi-mode Microplate Reader with OD Ex:488 Em:525. \*\*\*
indicates *p* < 0.001.

Furthermore, we calculated the On/Off ratios of LIRA with different
stem-loop ratio, i.e., H142\_3/20, H142\_8/15 and H142\_11/12, and found
that H142\_11/12 has the biggest On/Off ratio, indicating that 11/12
might be the best stem-loop ratio for LIRA that targets miR-142-3p.

![](https://static.igem.wiki/teams/5038/img-results/15.png)

**Figure 15**. On/Off fluorescence of H142 in stem-loop ratio of
3/20, 8/15 and 11/12.

Test of single-arm LIRA that targets miR-210-3p

Our model group also designed single-arm LIRAs that target miR-210-3p,
we chose 3 LIRAs with different stem-loop ratios for evaluation. We
cloned these LIRAs into pCOLADuet-1, and named these constructs
H210\_2/20, H210\_7/15 and H210\_10/12 respectively. In addition, we cloned
the sequence of miR-210-3p into plasmid pCDFDuet-1 and pET-15b for the
expression of miR-210-3p, and named these constructs input210.

**Table 2**. Plasmid for H210.

| Number | Plasmid description | Device |
| --- | --- | --- |
| 1 | pCOLADuet-H210\_2/20-linker-EGFP | PT7-LIRA\_H210\_2/20-linker-EGFP-T7TE |
| 2 | pCOLADuet-H210\_7/15-linker-EGFP | PT7-LIRA\_H210\_7/15-linker-EGFP-T7TE |
| 3 | pCOLADuet-H210\_10/12-linker-EGFP | PT7-LIRA\_H210\_10/12-linker-EGFP-T7TE |
| 4 | pCDFDuet-input210 | PT7-LIRA\_input210-T7TE |
| 5 | pET-15b-input210 | PT7-LIRA\_input210-T7TE |

All plasmids listed in Table 2 were transformed into BL21-DE3 (Figure
16) and plasmids from successfully transformed clones were verified by
digestion with restriction endonuclease and agarose gel electrophoresis
(Please see our [Engineering](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D) section for detailed information).

![](https://static.igem.wiki/teams/5038/img-results/16.png)

**Figure 16**. Plate for H210 related transformation.

Transformed BL21-DE3 clones was treated with 0.1mM IPTG for 4h and then
was disrupted by ultrasonic-sound. Cell lysate was collected by
centrifuging and diluted to 1.8 μg/μL. The EGFP fluorescence from the
diluted cell lysate was measured at a medium-high PMT gain. The results
showed that the clones transformed with H210\_2/20 and input210 have
highest expression of EGFP than other clones.

![](https://static.igem.wiki/teams/5038/img-results/17.png)

**Figure 17**. Fluorescence comparison of BL21-DE3 with H210 related
plasmids by Multi-mode Microplate Reader with OD Ex:488 Em:525. \*\*\*
indicates *p* < 0.001.

Furthermore, we calculated the On/Off ratios of LIRA with different
stem-loop ratio, i.e., H210\_2/20, H210\_7/15 and H210\_10/12, and found
that H210\_2/20 has the biggest On/Off ratio, indicating that 2/20 might
be the best stem-loop ratio for LIRA that targets miR-210-3p.

![](https://static.igem.wiki/teams/5038/img-results/18.png)

**Figure 18**. On/Off fluorescence of H210 in stem-loop ratio of
2/20, 7/15 and 10/12.

Test of double-arm LIRA that target both
 miR-142-3p and miR-210-3p

In this stage, our goal was to verify the effectiveness of our designed
double-arm LIRA that target both miR-210-3p and miR-142-3p. The
double-arm LIRA has two recognition regions for two different target
RNA, and an ideal LIRA could be activated only in the presence of both
these two target RNA. Our model group analyzed the sequence and
structure of LIRA, and used multiple indicators to calculate the
expectation score of LIRA to predict its effectiveness (Please see our
[Model](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D) section for detailed information). We chose 3 double-arm LIRA with
different expectation scores to test their effectiveness on detecting
miR-210-3p and miR-142-3p.

We cloned these LIRAs into pCOLADuet-1, and named these constructs
double-arm LIRA 1, double-arm LIRA 2, and double-arm LIRA 5
respectively.

**Table 3**. Plasmid for double-arm LIRA.

| Number | Plasmid description | Device |
| --- | --- | --- |
| 1 | pCOLA-PT7-LIRA\_ANDgate1-linker-EGFP-T7TE | PT7-LIRA\_ANDgate1-linker-EGFP-T7TE |
| 2 | pCOLA-PT7-LIRA\_ANDgate2-linker-EGFP-T7TE | PT7-LIRA\_ANDgate2-linker-EGFP-T7TE |
| 3 | pCOLA-PT7-LIRA\_ANDgate5-linker-EGFP-T7TE | PT7-LIRA\_ANDgate5-linker-EGFP-T7TE |
| 4 | pET-15b-input142 | PT7-LIRA\_input142-T7TE |
| 5 | pCDFDuet-input210 | PT7-LIRA\_input210-T7TE |

All plasmids listed in Table 3 were transformed into BL21-DE3(Figure
19-21) and plasmids from successfully transformed clones were verified
by digestion with restriction endonuclease and agarose gel
electrophoresis (Please see our [Engineering](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D) section for detailed
information).

![](https://static.igem.wiki/teams/5038/img-results/19.png)

**Figure 19**. Plate for pCOLA-LIRA\_ANDgate1-linker-EGFP related
transformation (BL21-DE3). From left to right. Plate1:
pCOLA-LIRA\_ANDgate1-linker-EGFP selected by Kan.

Plate2: pCOLA-LIRA\_ANDgate1-linker-EGFP+pET-15b-input142 selected by Kan
and Amp.

Plate3: pCOLA-LIRA\_ANDgate1-linker-EGFP+pCDFDuet-input210 selected by
Kan and STC.

Plate4:
pCOLA-LIRA\_ANDgate1-linker-EGFP+pET-15b-input142+pCDFDuet-input210
selected by Kan and Amp and STC.

![](https://static.igem.wiki/teams/5038/img-results/20.png)

**Figure 20**. Plate for pCOLA-LIRA\_ANDgate2-linker-EGFP related
transformation (BL21-DE3). From left to right. Plate1:
pCOLA-LIRA\_ANDgate2-linker-EGFP selected by Kan.

Plate2: pCOLA-LIRA\_ANDgate2-linker-EGFP+pET-15b-input142 selected by Kan
and Amp.

Plate3: pCOLA-LIRA\_ANDgate2-linker-EGFP+pCDFDuet-input210 selected by
Kan and STC.

Plate4:
pCOLA-LIRA\_ANDgate2-linker-EGFP+pET-15b-input142+pCDFDuet-input210
selected by Kan and Amp and STC.

![](https://static.igem.wiki/teams/5038/img-results/21.png)

**Figure 21**. Plate for pCOLA-LIRA\_ANDgate5-linker-EGFP related
transformation (BL21-DE3). From left to right. Plate1:
pCOLA-LIRA\_ANDgate5-linker-EGFP selected by Kan.

Plate2: pCOLA-LIRA\_ANDgate5-linker-EGFP+pET-15b-input142 selected by Kan
and Amp.

Plate3: pCOLA-LIRA\_ANDgate5-linker-EGFP+pCDFDuet-input210 selected by
Kan and STC.

Plate4:
pCOLA-LIRA\_ANDgate5-linker-EGFP+pET-15b-input142+pCDFDuet-input210
selected by Kan and Amp and STC.

Transformed BL21-DE3 clones was treated with 0.1mM IPTG for 4h and then
disrupted by ultrasonic-sound. Cell lysate was collected by centrifuging
and diluted to 1.8 μg/μL. The EGFP fluorescence from the diluted cell
lysate was measured at a medium-high PMT gain. The clones transformed
with double-arm LIRA 1 did not have increased EGFP expression even with
both input142 and input210, indicating that it could not be activated by
miR-142-3p and miR-210-3p (Figure 22). The clones transformed with
double-arm LIRA 2 showed increased EGFP expression even with input142
alone, indicating that it could be activated by miR-142-3p alone (Figure
23). The clones transformed with double-arm LIRA 5 showed increased EGFP
expression only with both input142 and input210, indicating that it
could only be activated in the presence of both miR-142-3p and
miR-210-3p (Figure 24).

![](https://static.igem.wiki/teams/5038/img-results/22.png)

**Figure 22**. Fluorescence comparison of BL21-DE3 with Double-arm
LIRA 1 related plasmids by Multi-mode Microplate Reader with OD Ex:488
Em:525.

![](https://static.igem.wiki/teams/5038/img-results/23.png)

**Figure 23**. Fluorescence comparison of BL21-DE3 with Double-arm
LIRA 2 related plasmids by Multi-mode Microplate Reader with OD Ex:488
Em:525. \*\*\* indicates *p* < 0.001.

![](https://static.igem.wiki/teams/5038/img-results/24.png)

**Figure 24**. Fluorescence comparison of BL21-DE3 with Double-arm
LIRA 5 related plasmids by Multi-mode Microplate Reader with OD Ex:488
Em:525. \*\*\* indicates *p* < 0.001.

Test of cell-free system

In the future, we would like to apply our LIRA-based screening approach
to screen blood samples from post-MI patients, to identify those
patients with high risk of cancer. Since it is unlikely that we could
transform miRNAs from blood samples into bacteria, we need to adapt our
LIRA-based screening approach into a cell-free system. In addition, for
a screening approach, it would be much easier to detect changes of color
than expression of EGFP, which requires machine to measure fluorescence.
Therefore, we set to make two improvements of our LIRA-based screening
approach. First, we change the reporter gene from EGFP to LacZ, which
could hydrolyze CPRG (yellow) into CPR (purple)[2]. Second,
we use a cell-free system to express the LacZ cloned in our LIRA
constructs.

To set up the cell-free system for LIRA, we first tried H01 LIRA that we
have successfully validated in our experiments at the beginning. We
replaced the reporter gene from EGFP to LacZ in H01 LIRA construct. For
the cell-free system, we chose the PURExpress® In Vitro Protein
Synthesis from New England BioLabs. With CPRG as substrate, the results
of LacZ expression could be visualized. (Please see our [Engineering](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D)
section for detailed information)

Next, we combined our double-arm LIRA 5 with a LacZ reporter gene, and
tested it with the cell-free system. We added the double-arm LIRA 5
plasmid with miRNA and CPRG into the cell-free system. After incubation
at 37 ° C for 2h, the results could be visualized, and also quantified
by measuring OD562 with a Multi-mode Microplate Reader. The
result demonstrated that our double-arm LIRA 5 could be activated by
miR-142-3p and miR-210-3p in the cell-free system (Figure 25).

![](https://static.igem.wiki/teams/5038/img-results/25-2.png)

**Figure 25** OD562 measurement results. Tube 1:
pCOLA-LIRA\_ANDgate5-linker-EGFP in cell-free system with CPRG. Tube 2:
pCOLA-LIRA\_ANDgate5-linker-EGFP and miR-142-3p in cell-free system with
CPRG. Tube 3: pCOLA-LIRA\_ANDgate5-linker-EGFP and miR-210-3p in
cell-free system with CPRG. Tube 4: pCOLA-LIRA\_ANDgate5-linker-EGFP,
miR-142-3p and miR-210-3p in cell-free system with CPRG. Tube 5: β-gal
with CPRG. \*\*\* indicates *p* < 0.001.

Reference

[1] Ma D, Li Y, Wu K, et al. Multi-arm RNA junctions encoding molecular
logic unconstrained by input sequence for versatile cell-free diagnostics.
Nat Biomed Eng. 2022;6(3):298-309.

[2] Ma D, Shen L, Wu K, Diehnelt CW, Green AA. Low-cost detection of
norovirus using paper-based cell-free systems and synbody-based viral
enrichment. Synth Biol (Oxf). 2018;3(1):ysy018.

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