iGEM 2023 — Team nthu-taiwan

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{% block title %}Diversity & Inclusivity{% endblock %}
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Notebook

* [February](#February)
* [March](#March)
* [April](#April)
* [May](#May)
* [June](#June)
* [July](#July)
* [August](#August)
* [September](#September)
* [October](#October)

# February

# Week 1

WL:

Wet Lab group were divided into four groups. To seek
inspiration for our project, each group is tasked with researching and reporting on previous
award-winning iGEM teams.

HP:

1. Project brainstorming and previous case studies.
2. Ensure scholarship deadlines and apply requirement.

# Week 2

WL:

We were still studying the past winning projects and tried to derive possible themes from their projects. In this week, four proposals emerged.

HP:

1. Brainstorm Projects and previous case studies.
2. Ensure scholarship deadlines and apply requirement.

# Week 3

WL:

1. We attempted to identify more promising projects.
2. Consequently, four additional new proposals came to fruition this week.

HP:

1. Brainstorm Projects and previous case studies.
2. Ensure scholarship deadlines and apply requirement.

# Week 4

WL:

We provided more detailed redesigns for four out of the
eight proposals discussed in the last two weeks. Following this, we had a discussion to
determine their feasibility.

HP:

1. Brainstorm Projects and previous case studies.
2. Ensure scholarship deadlines and apply requirement.

# March

# Week 1

WL:

In this week, the WL team was divided into two groups
instead
of four. Each group had to choose one out of the four proposals discussed last week and delve
into
further discussions. We faced a difficult decision between 'disease detection' and
'bio-electricity'. In the end, we opted to pursue 'bio-electricity' as our subject, aiming to
generate electricity through microorganisms.

DL:

Discuss the application of microbial fuel cell and to think
up an innovative solution to apply.

HP:

1. Contact entrepreneurs and inquiry of our project
suggestion.
2. Explan different thoughts of biodegradable materials for
kit's
cover.
3. Discuss online educational program.
4. Design advertisement music.
5. Apply and prepare of current scholarships.

# Week 2

WL:

We came up with two ideas related to bio-electricity.
Firstly, we aimed to desalinate seawater using a microbial fuel cell. Secondly, we proposed to degrade
polylactate, synthesized by E. coli, into lactate using an optogenetic system. The lactate would
then be utilized to generate electricity through Shewanella bacteria.

DL:

Understand the working principle of detection kit and find
the appropriate professors to help. Also determine the structure of Dry Lab.

HP:

1. We discussed an online educational program.
2. We designed advertisement music.
3. Apply and prepare for current scholarships.

# Week 3

WL:

After conducting extensive data querying, we discovered
that the development of microbial fuel cells has reached an advanced stage. Consequently, we shifted
our focus towards identifying promising proposals in the field of disease detection, which was the
right issue we tried to deal with.

DL:

Separate the job by individual strength. Also discuss about
using proper method to predict the structure.

HP:

1. We prepared posting scientific-related knowledge on an
Instagram account for promoting synthetic biology.
2. Discussed about comic book design.

# Week 4

WL:

1. We opted to utilize circular RNA present in human saliva as
a biomarker for cancer detection.
2. Tried to use several DNA oligo with hairpin structure to
recognized and amplify circular RNA.
3. Planned to employ a lateral flow strip to display the test
results.

DL:

Try to use KinDA to simulate the hairpins, while using the
3D RNA score to compare and select the most ideal target.

HP:

1. We prepared posting scientific-related knowledge on an
Instagram account for promoting synthetic biology.
2. We contact professors and inquiry of project direction and
further research methods.

# Week 5

WL:

1. We identified three circular RNA that highly related to
breast cancer.
2. After consulting with some professors, they advised us to
explore types of cancer other than breast cancer that are challenging to detect using current
diagnostic methods.
3. Discovered some isothermal amplification methods. Such as
rolling circle amplification(RCA), recombinase polymerase amplification(RPA), reverse
transcription
recombinase polymerase amplification (RT-RPA) and Loop-mediated Isothermal Amplification(LAMP).

HP:

Finish our project report power point file and prepared
meeting with professor meeting.

# April

# Week 1

WL:

Comprehended the mechanism and reaction temperature of RCA,
RT-RPA, RPA, and LAMP, respectively.

DL:

Choose the target circRNA and determine how to calculate
the reaction rate. Further discuss the detail about 3D RNA score and RPA model.

HP:

1. Scholarship apply schedule (platform researching and email
contact) arranged.
2. Educational project content preparation.
3. Preparation for interviewing doctors.
4. Preparation for interviewing professors.

# Week 2

WL:

1. Discovered PCRD, a lateral flow cassette we applied to
detect cancer.
2. We tried to explore the experimental protocols of RCA and
RT-RPA using circular RNA as a template. In the end, we couldn't discover any literature
mentioned how to amplify circular RNA through RT-RPA.

DL:

Need to change the target circRNA and solve the problems of
the reaction rate and 3D RNA score. Heating equipment needs to find a battery for heating.

HP:

1. We arranged the scholarship application schedule (platform
research and email contact).
2. We prepared the educational project content.
3. We prepared interviews with doctors.
4. We prepared to interview professors.

# Week 3

WL:

1. Since there were no references mentioned on how to amplify
circular RNA by RT-RPA, we came up with another solution to fix this problem. We planned to
transform circular RNA to complementary DNA utilizing reverse transcription, then amplify the
cDNA through RPA.
2. We found that ProtoScript II reverse transcriptase
exhibited higher efficiency. We were curious about its potential inclusion in the RCA process.
3. Investigated the mechanism of PCRD, then determined its
feasibility.

DL:

Building up the models of RPA/ RCA/ Lamp, PCRD/ Hairpin and
fluid. Also establishing the software and wiki structure.

HP:

1. Team logo design.
2. Explanation about who the sponsors are.

# May

# Week 1

WL:

Investigated the MgCl2 concentration causing the
aggregation of RCA products, and determine the presence of modified probes or hairpin structures
on gold nanoparticles.

DL:

Check the sequence that cDNA is replacing. Also
establishing the buffer diffusion model. Then use software to judge the reaction is completed.

HP:

1. Discussion and preparation of an academic conference.
2. Grant prize apply—preparation and check.
3. Voting for the final decision of team logo design.

# Week 2

WL:

1. We found that after the circularization process, RNase R is
needed to eliminate linear form RNA. By adding RNase R, we could ensure the purity of our
circularization product.
2. Utilize selected circRNA as our target, design a
corresponding RCA primer.

DL:

Need to set up a terminal time for the diffusion model to
stop. The heating equipment needs to combine with software.

HP:

1. We designed our promotion video.
2. We discussed and contacted the biotechnological companies
and sponsors.
3. We contacted the travel agency and inquired about the
travel schedules.
4. We prepared our questionnaire.

# Week 3

WL:

1. Ensure whether the sequence in circBank is complementary to
circRNA or cDNA of circRNA.
2. Design a primer for RPA and RCA.
3. Design a DNA probe which is attached to a gold particle.

DL:

PCRD model needs to wait for the input value to be tested
and also think up the storing method and look up the changing color trend. For hardware,
designing the automatic placement of the salt.

HP:

1. Educational science camp preparation and discussion.
2. Inviting foreign and other iGEM teams for iGEM map
collaboration.

# Week 4

WL:

1. Ensure all the chemicals and kits are needed. Check
protocols and detailed information for further experiments.
2. Find access to thiol-modified DNA, then find protocols for
attaching thiol-modified DNA to gold nanoparticles.

DL:

List out the exact components for heating equipment. Think
up the tool for creating the application. Apply for the sponsorship or tuition.

HP:

1. Voting for the final decision of team logo design.
2. Wiki preparation and suggestion.

# June

# Week 1

WL:

1. Clarify cloning process, consider enzyme restriction site
for digesting insert and vector sequence.
2. Clarify each step for enzymatic circularization, including
the verification way using gel electrophoresis method.

DL:

Use Simscale for the diffusion model. Get familiar with
RCA, RPA, DnaProbe, and the PCRD model. Ask senior about the details of the heating equipment.

HP:

1. Wiki designing & concept brainstorming.
2. Wiki characters design.

# Week 2

WL:

1. Comparing the pros and cons of T4 RNA ligase 1 and T4 RNA
ligase 2. Determine using splint to bring the sequence close together, elevate circularization
efficiency.
2. Determine to use pyrophosphate in the monophosphorylation
process in circRNA synthesis.

HP:

1. Wiki designing & concept brainstorming.
2. Wiki characters design.

# Week 3

WL:

1. Design a splint for circularization. According to research,
we found that RNA might not spontaneously form a circular structure, thus we design a splint
targeting back-splicing junction for higher specificity.
2. Use RNAfold web server to predict the splint structure.
Avoid secondary structure of splint, also design the entropy of the splint close to 1.

HP:

1. Preparation and discussion of educational science camp and
life science camp promotion.
2. Shoot photos for each lab.

# Week 4

WL:

1. We found that we should eliminate redundant splint DNA
after circularization. So we determine to use DNase I for degradation of splint DNA.
2. Determine the type of gel we used to verify the
circularization process. Candidates including polyacrylamide gel, agarose gel. Find the pros and
cons of each type of gel.
3. Determine the vector we're going to use in the cloning
project.

DL:

Reaching the tutorial of Simbiology. Briefly setting up the
geometric factors of the diffusion model. Wiki had added the progress list and the sidebar.

HP:

1. Making our own questionnaire.
2. Preparation for NYCU academic conference communication.

# Week 5

WL:

1. We tried to find previous research related to developing
long-repeated ligased insert sequence in AELA-PCR.
2. Ensure the condition to ligase splint to our target
sequence for circularization.

HP:

We made the final decision of the traveling agency and
traveling schedule.

# July

# Week 1

WL:

1. Clarify each step in our workflow pathways.
2. Try to synthesize gold nanoparticles.

DL:

1. Understand that the radius may affect the segregation
extent. Purchase the essential components and discuss the possible game type to create.
2. Discuss the game design thoughts with Human Practice.
Determine the game content, background and how to combine the information in it.

HP:

1. Collaboration response of World Nature Conservation Day.
2. Wiki character design progress.
3. Colorectal Cancer alliance contact and biotech companies
contact.
4. Preparation of educational science camp.
5. Preparation for flight to France.

# Week 2

WL:

1. Clarify condition of protocols in our workflow pathways.
2. Confirm rotating speed of hula mixer and stir bar with
advisor.

DL:

Understand the reasons of segregation and changing color,
further quantify the extent. Add some word effects in wiki page.

HP:

1. Wiki character design progress.
2. Preparation for interviewing Colorectal Cancer's patient
and doctor.
3. Research for funding platform.
4. Preparation for online meeting collaboration.
5. Instagram post of educational content brainstorming.
6. Scholarship and sponsors contact.
7. Wiki page and report model design.

# Week 3

WL:

1. Clarify condition of protocols in our workflow pathways.
2. Doing research and making comparisons of running circRNA in
E-Gel EX and Agarose gel.
3. We found that band position of linear RNA and circRNA have
difference in E-Gel EX, we could use the difference to distinguish linear RNA from circRNA.

DL:

Have solved the problems of heating equipment. Try to use
machine learning for setting up the model. Improving the game fluency.

HP:

1. Wiki character design progress.
2. Preparation for interviewing Colorectal Cancer's patient
and doctor.
3. Research for funding platform.
4. Preparation for online meeting collaboration.
5. Instagram post of educational content brainstorming.
6. Scholarship and sponsors contact.
7. Wiki page and report model design.

# Week 4

WL:

1. Clarify condition of protocols in our workflow pathways.
2. Try to synthesize gold nanoparticles, modified O.D. value
by centrifuge synthesized gold nanoparticle samples.

DL:

Can use Simbiology to generate ODEs and simulate the graph.
The blueshift range has determined. Renewing the games and thoughts on the wiki page.

HP:

1. Instagram reels collaboration of lab life.
2. Team roster check.
3. Mentorship contact and problem solving.
4. Wiki graphic preparation.
5. Traveling agency contacting.
6. Apply for current scholarship.

# August

# Week 1

WL:

1. We prepared the needed buffer for gold nanoparticle
synthesis in advance.
2. Senior Peter took us to combine nanogold + probe on 8/5.
3. Prepare the report and poster of the Linkou exchange
meeting.

DL:

Collaborate with several teams, prepare the script and
rehearse beforehand. Thinking up the structure of application for machine learning.

HP:

1. Preparation and discussion for promotion video.
2. Discussion of wiki page design.
3. Preparation of NYCU educational podcast collaboration and
Instagram reels collaboration.
4. Preparation of educational science camp work sheets.
5. Contact for entrepreneurship interview.

# Week 2

WL:

1. Rehearsal for the Linkou exchange conference.
2. Read carefully about judging criteria, judging form from
iGEM.

DL:

Rehearse for the upcoming collaboration and giving some
possible problems. Revise the script to improve fluency.

HP:

1. Preparation and discussion for promotion video.
2. Discussion of wiki page design.
3. Preparation of NYCU educational podcast collaboration and
Instagram reels collaboration.
4. Preparation of educational science camp work sheets.
5. Contact for entrepreneurship interview.

# Week 3

WL:

1. Prepare for the education camp and decide how to convey the
synthetic biology concept to children so they can understand.
2. Conducting ligation with vector experiment part in
AELA-PCR, we tried to endlong the time for better ligation outcome.
3. Organize wiki structure and content.

DL:

Rehearse for the collaboration. Finding the relationship of
cDNA radius to the segregation of the particles. Put the collaboration team by geographical
location in wiki page.

HP:

1. Wiki background design and wiki game progress report.
2. Discussion for team cloth design.
3. Preparation for entrepreneur intervie with doctor interview.
4. Final check of NYCU academic conference communicational
preparation and discussion.
5. Preparation for educational science camp.
6. Preparation and final checking of scholarship applying
document.

# Week 4

WL:

1. We successfully synthesized 13nm AuNPs.
2. We found out that EDS cannot detect the probe, so we seek
to use XPS to assist in the detection of probes conjugated with AuNPs.
3. We conducted RT-RPA experiment, however failed due to no
template control shows band.
4. Conduct Insert PCR, and colony PCR.

DL:

Discuss about the volcano plot, distribution of particles
and calculate the segregate extent. Had added the reset button, music, etc. in the game.

HP:

1. Explanation of project related SDGs.
2. Questionnaire form discussion.
3. Advices and suggestions explaining for presentation
methods.
4. Judging rubric explanation.
5. Wiki game progress report.
6. Reminder for Michigan Synthetic Biology team collaboration.
7. Preparation for traveling.

# Week 5

WL:

1. We saw 13nm gold nanoparticle under XPS.
2. We diluted the RT-RPA sample and run gel to see the testing
limit of the amplification method.

DL:

Determine the necessity of AUC/ ROC graphs. Simulate the
diffusion phenomenon in several ways. Redesign the title and add maps of wiki page.

HP:

1. Animation making of experimental process of RCA, RPA, nano
particles gathering, circular RNA detecting and wiki game.
2. Preparation and discussion of promotion video timeline.

# September

# Week 1

WL:

1. We discussed safety form content, and finished them.
2. We conduct circularization experiment. we analyzed and
reviewed problems which may lead to circularization experiment failure.
3. We conduct AELA experiment. We successfully finished the
parts including insert ligation, digestion, ligation with vector, however failed at the process
of transform.
4. We prepared a waiver for the gold nanoparticle we have
synthesized.
4. We conduct RT-RPA experiment. Unfortunately, we failed the
experiment since there is always a band in no template control (NTC). We tried to analyze and
review the problems in each step which may lead to RT-RPA experiment failure.
5. We successfully conducted RPA experiment to ensure the RPA
primer we designed works.

DL:

Add some effects in the game and discuss the ranking
system. The hardware simulation graph has been elaborated.

HP:

1. Completing scholarship applying document.
2. Discussion of presentation way for NYCU academic conference
communication.
3. Reminder of promoting synthetic biology on science camp.

# Week 2

WL:

1. Try the condition of adding how much MgCl2 will lead to
gold nanoparticle solution color change.
2. Referring Wiki page from the 7th-grade NTHU iGEM team.
3. We found out that there should be a minimum concentration
of IVT concentration before conducting the circularization experiment.
4. We successfully conduct the circularization experiment.

DL:

Can use R language to run the cluster heat map. The
revision on hardware equipment.

HP:

1. Checking for scholarship applying document.
2. Explanation and discussion of wiki game transcription.
3. Presentation video editing.

# Week 3

WL:

1. Starting the blue print of our wiki design-Experiment.
2. Starting the blueprint of our wiki design-Proof of Concept.
3. Starting the blueprint of our wiki design-Engineering
Cycle.
4. Revising our reagents used for the RCA experiment, by
changing ProtoScript II RT to Induro, since we couldn't get multiple copies of cDNA.

DL:

Update the wiki page. Revise the equations of the PCRD
model and transform the equations into ODEs. Design a handbook of the hardware. Need some
pictures for the PCRD model for machine learning.

HP:

1. Completing promotion video shooting, editing, and
uploading.
2. Preparation of traveling schedule.

# Week 4

WL:

1. Revising the process of the Circularization experiment to
get a better yield of the target circularized RNA.
2. Referenced the iGEM Wiki of three other schools.
3. Decide how to express our Wiki design-Proof of Concept.
4. Decide how to express our Wiki design-Engineering cycle by
dividing into 3 main sections.

DL:

Confirming and retrieving the data with Wet Lab, further
integrate the model. Upload the code onto github and renovate the hardware.

HP:

1. Writing wiki content for human practice pages and related
graphic drawing.
2. Final check for scholarship applying document.
3. Report for traveling schedule.

# October

# Week 1

WL:

1. Wiki content submission: continue modifying Wiki layout,
Wiki content.
2. Uploading experiment pictures and diagrams onto Wiki.

DL:

Using the initial value from paper. Need to set up a
time-depending function for diffusion. Do the animation of the whole automatic procedure. Debug
the wiki page.

HP:

1. Checking and modifying the judging form.
2. Wiki content completing and discussion.

# Week 2

WL:

1. We successfully conduct the RT-RPA experiment with no band
showing on the no template control line.
2. We finished Wiki content based on the wet labs part.

DL:

Finish up the application, machine learning model. Do the
diffusion model and compare under different concentrations.

HP:

1. Preparation and discussion of the presentation video.
2. Shooting and editing the presentation video.

# Week 3

HP:

1. Preparation for France traveling.
2. Preparation for final presentation and questions.

# Week 4

HP:

1. Preparation for France traveling.
2. Preparation for final presentation and questions.

# Week 5

HP:

Fly to France.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering{% endblock %}
{% block page\_content %}

Engineering

* [Introduction](#Introduction)
* [Section 1](#Section 1)

+ [Cycle 1](#Cycle 1)
+ [Cycle 2](#Cycle 2)

* [Section 2](#Section 2)

+ [Cycle 1](#Section 2Cycle 1)
+ [Cycle 2](#Section 2Cycle 2)

* [Section 3](#Section 3)

+ [Cycle 1](#Section 3Cycle 1)
+ [Cycle 2](#Section 3Cycle 2)

* [Section 4: model](#Section 4)

+ [Cycle: Difussion](#Section 4Cycle : Difussion)

* [Section 5: model](#Section 5)

+ [Cycle: Amplification](#Section 5Cycle : Amplification)

* [Reference](#Reference)

# Introduction

When it comes to synthetic biology,
it is important to ensure that the results of our project align with our expectations. However,
before we successfully achieve our desired goals, there is a long way to go due to the
unpredictability of the results. For this situation, we introduce an important principle: the
engineering cycle, often abbreviated as the DBTL (Design, Build, Test, and Learn) cycle, to
divide the entire experiment into four sections. The DBTL cycle helps us pinpoint where
potential issues may arise more easily. When it goes wrong, we can redesign and rebuild a cycle
to lead our biological system in the right direction and finally reach its destination. Beyond
the four stages in the DBTL cycle, we include a 'recap' section after the learning stage to
summarize actions for enhancing our experiment.

![](https://static.igem.wiki/teams/4636/wiki/engineering-success/figure-1.jpg)

Figure 1. Engineering cycle.

# Section 1: Enhancing the yields of circular RNA (circRNA).

# Cycle 1

# Design:

In the attempt to
circularizing linear RNA, we designed a DNA oligo, known as splint. The splint has a
complementary sequence to the linear RNA. With this characteristic, it enhances the
circularization ability of our RNA, by forming a double-stranded RNA (Figure 2) to circularize
RNA. After the ligation process is done, the splint will be degraded by treating with DNase I.
(See [page design](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27design%27%29%20%7D%7D) for the design of circularization). [[1]](#[1])

![](https://static.igem.wiki/teams/4636/wiki/engineering-success/figure-2.png)

Figure 2. A diagram shows how
splint helps the formation of circular RNA.

# Build:

In the very beginning, we only
noticed the basic principle of splinting. Such a splint needs to be a complementary sequence to
linear RNA that acts as a platform that can ligate the ends of RNA, helping it to form
the covalently-closed loop structure. Initially, we thought that the longer a complementary
splint, the better its ligation ability, so we designed splint sequences ranging from 12 to 30
base pairs long. However, there would be too many candidates (10 exactly) if we designed and
produced all the splints, which means a higher cost would be required, and this was not
possible. Thus, we took a previous study [[2]](#[2]) into account. The paper mentions a method for
circularizing DNA from its linear form. However, this is the closest idea aligning with our
project, so we had no option but to take their previous experience and parameters into
consideration. In this study, it said that the best circularization efficiency is at 12 bp,
nevertheless, the Tm value of our splint with 12bp was too low for the reaction. As a result, we
mimicked their experiment and decided to design a 14 bp splint, Splint\_1802\_14 (BBa\_K4636015)
and Splint\_4771\_14 (BBa\_K4636018) for circularizing RNA. (See [page parts](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27parts%27%29%20%7D%7D) for detail
information)

# Test:

Here only shows the result of
circularization using Splint\_1802\_14. To make sure the products of each step in synthesizing
circRNA (See [page design](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27design%27%29%20%7D%7D) for details) were produced as expected, we decided to sequentially
verify the effectiveness of each step and conduct E-Gel EX gel electrophoresis tests. Firstly,
the presence of a band at lane 1 indicated a successful in vitro transcription (IVT) process, as
we treated the reagent with DNase I. Secondly, we added XRN-1, a chemical capable of degrading
monophosphorylated RNA [[3]](#[3]) (lane 2: Without XRN-1, lane 3: With XRN-1 added). Lastly, given the
circular form of RNA's resistance to RNA exonuclease activity, we could verify the yields in the
back-splicing experiment by treating them with RNase R [[4]](#[4]). Similarly, we also treated the
back-splicing product with XRN-1 in an attempt to degrade monophosphorylated linear RNA for
further validation. (Lane 4: Without RNase R; lane 5: With RNase R added; lane 6: With XRN-1
added).

![](https://static.igem.wiki/teams/4636/wiki/engineering-success/figure-3.jpg)

Figure 3. Lane 1: IVT product, Lane
2: Monophosphorylation product, Lane 3: Monophosphorylation product + XRN-1, Lane 4:
Circularization product, Lane 5: Circularization product + RNase R, Lane 6: Circularization
product + XRN-1

# Learn:

From the result of E-Gel EX
electrophoresis, lane 1, 2, and 3 displayed the outcomes we anticipated. In other words, in
vitro transcription and monophosphorylation were confirmed to be successful. However, lanes 4,
5, and 6 exhibited such shallow bands that they were almost imperceptible. This indicated a low
yield in our back splicing procedure.

# Recap:

Knowing that the failure came from
circularization, we then tried to search for more resources to explore any detail we might have
ignored before.

# Cycle 2

# Design:

After looking through papers and
experiments done by previous people, we realized that the ligation efficacy of circular RNA
depends on the CG content, the structure, the Tm value, and the entropy value. [[5]](#[5]) Besides, we
also noticed that the Tm value is a cofactor that affects the ligation ability. Splint's Tm
value should be more than 25°C, so that it would be able to combine with the DNA more firmly.
Last but not least, we thought of testing our designed splint sequence to predict entropy value
and structure by running through a website [[6]](#[6]). We consider that the reason why our previous
circularization experiment with 14 base pair splint didn't succeed in circularizing RNA lies in
two main reasons: Tm value and GC content.

For the 14bp splint (For linear RNA
of Insert\_0101802, BBa\_K4636040), the Tm value was lower than the 16bp splint, which means it
may not correctly bind to our target RNA since the Tm of the splint should be higher than 25 °C
to make it anneal with the DNA more firmly.

While for GC content, 14bp splints
are 50% and 16bp splint are 43.8%. Since the higher GC%, the more difficult it will be to
unscrew the double helix, the 14bp splint may have relatively poor ability compared with the
16bp splint.

The reason mentioned above and the
previous 14bp experiment failure made us want to try a longer splint length, so we designed two
more splint lengths, which were 16 and 18 base pairs long.

# Build:

We finally build our splint
sequence through filter criteria mentioned above, like CG content, avoiding secondary structure,
splint Tm, and entropy values. This also included conducting the previous paper, so we designed
two more different lengths, which were 16 (Splint\_1802\_16 ,BBa\_K4636014) and 18
(Splint\_1802\_18, BBa\_K4636013) base pairs long.

# Test:

To prove the success of our new
design, circularization product (With RNase R added) using Splint\_1802\_14 (BBa\_K4636015)
, Splint\_1802\_16 (BBa\_K4636014) and Splint\_1802\_18 (BBa\_K4636013) were tested together using
agarose gel electrophoresis.

![](https://static.igem.wiki/teams/4636/wiki/engineering-success/figure-4.png)

Figure 4. Lane 1: low range ssRNA ladder. Lane 2: Negative control. Lane 3: Back-splicing
product using Splint\_1802\_14. Lane 4: Back-splicing product Splint\_1802\_16. Lane 5:
Back-splicing product Splint\_1802\_18.

# Learn:

Based on the result of agarose gel
electrophoresis (Figure 4), we could identify that the splint with 16bp and 18bp have higher
back-splicing efficiency than 14bp as we expected.

# Section 2: Identify the optimal conditions for RCA

# Cycle 1

# Design:

Since both the circular RNA we
selected present low concentration in the human serum, we choose to amplify them through an
isothermal amplification method called rolling circle amplification (RCA) (see [page design](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27design%27%29%20%7D%7D) for
details).

![](https://static.igem.wiki/teams/4636/wiki/engineering-success/figure-5.jpg)

Figure 5. A diagram shows how RCA amplify circRNA by producing long repeated single-stranded
complementary DNA.

# Build:

Followed by paper studying, the
Protoscript II reverse transcriptase (New England BioLabs) seems to be the the most commonly
used enzyme when conducting RCA using circRNA as template [[7]](#[7]). Therefore, we decided to use
Protoscript II reverse transcriptase to amplify our target biomarkers and conducted agarose gel
electrophoresis to present the result.

# Test:

In theory, reverse transcriptase
can repetitively amplify the circRNA to produce a long-repeated single-stranded DNA (ssDNA)
(Figure 5). Conversely, the linear RNA can only be reverse transcribed into complementary DNA
(cDNA) with a length identical to that of the RNA template. Consequently, we conducted RCA using
both linear and circular RNA to evaluate the effectiveness of RCA.

![](https://static.igem.wiki/teams/4636/wiki/engineering-rtrpa/engineering-6.png)

Figure 6. Lane1: ssRNA ladder. Lane
2: RCA product using circularized RNA as template. Lane 3: RCA product using linear RNA as
template.

# Learn:

From the result of agarose gel
electrophoresis (Figure 6), though the single-stranded cDNA was successfully synthesized, the
length remained the same as the linear form of RNA even though the reaction time was extended.
In this case, we assumed that there might be some inherent limitations to protoscript II reverse
transcriptase.

# Recap:

To deal with the question we
hypothesized, all of us spared no effort to collect and integrate related literatures.

# Cycle 2

# Design:

After extensive data querying, we
found that the amplification ability of Protoscript II reverse transcriptase is limited to
around 500 base pairs. On the other hand, we also discovered a promising reverse transcriptase
called Induro® Reverse Transcriptase (New England BioLabs)with an amplification ability of
around 12,000 base pairs [[8]](#[8]).

# Build:

We followed the protocol provided
by the manufacturer. Since the manufacturer suggested a reaction time between 15-30 minutes, we
conducted the reaction with durations of 15 and 30 minutes respectively to determine the optimal
condition.

# Test:

We used a ssRNA ladder (New England
BioLabs) (lane 1) to recognize the length of our reaction product. To determine the optimal
reaction time, we loaded RCA products using circular RNA (circRNA) as a template with reaction
times of approximately 15 minutes (lane 2) and 30 minutes (lane 3), respectively. RCA products
using linear RNA with the RNA concentration remaining the same as in lane 2 and 3 were used as
templates. Both reaction times of 15 minutes (lane 4) and 30 minutes (lane 5) were tested. For
lane 6 and lane 7, the components were essentially the same as in lane 4 and lane 5, with the
only difference being that the RNA concentration was 10 times higher. (Figure 7)

![](https://static.igem.wiki/teams/4636/wiki/engineering-success/figure-7.png)

Figure 7. Lane 1: ssRNA ladder. Lane 2: CircRNA as template with reaction time of 15mins. Lane3:
CircRNA as template with reaction time of 30 mins. Lane 4: Linear RNA with the concentration
identical to lane 2 and lane 3 as template and the reaction time of 15mins. Lane 5: Linear RNA
with the concentration identical to lane 2 and lane 3 as template and the reaction time of
30 mins. Lane 6: Linear form RNA with concentration 10 times higher and the reaction time of
15mins. Lane 7: Linear form RNA with concentration 10 times higher and the reaction time of
30 mins.

# Learn:

According to the results (Figure
7), we observed that lane 2 and lane 3 displayed significantly larger RCA products
(approximately 1000 to 2000 nt) compared to lane 4 to lane 7 (approximately 450 nt). This
indicated that the Induro reverse transcriptase successfully amplified the RNA signal when
circRNA was used as the template.

# Section 3: Adjusting condition for probe-AuNPs

# Cycle 1

# Design:

For the colorimetric assay, we
need to attach DNA probes with sequence complementary to our RCA products on the surface of gold
nanoparticles (see page [description](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27description%27%29%20%7D%7D) for details of gold nanoparticle based-colorimetric assay) [[9]](#[9]).

![](https://static.igem.wiki/teams/4636/wiki/engineering-success/figure-8.jpg)

Figure 8. A diagram shows the
mechanism of gold nanoparticle based-colorimetric assay.

According to the literature, there
will be a redshift in wavelength when probes are attached [[10]](#[10]). Therefore, in this section, our
aim is to explore suitable experimental method to synthesize probe-conjugated gold nanoparticles
with red shift in wavelength.

# Build:

We referred to the protocol
provided in the literature and labeled it as Option 1 (see [page experiment](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D) for protocols) [[9]](#[9])..
Subsequently, we determined the redshift in wavelength using the nanodrop technique.

# Test:

![](https://static.igem.wiki/teams/4636/wiki/engineering-success/figure-9.jpg)

Figure 9. The comparison of wavelengths between the pure gold nanoparticle solution and solution after processing Option 1.

# Learn:

Based on Figure 9, we can observe
that there was no red shift in wavelength, which indicated we failed to attach probes on the
surface of gold nanoparticles.

# Recap:

We hypothesized the failure of our
attempt to synthesis probe-conjugated gold nanoparticles was a result of unsuitable experimental
conditions. Consequently, we tried to find an alternative method to produce the complex.

# Cycle 2

# Design:

Learned from the cycle 1, our
adviser suggested us to study the references [[11]](#[11]) provided by the previous paper to figure out if
there were other solution to synthesize the probe-conjugated gold nanoparticles we desired.

# Build:

Fortunately, we had found out
another protocol, named it Option 2 (see [page experiment](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D) for protocols) [[11]](#[11]), for conjugating
probes and gold nanoparticles. As mentioned in the cycle one, we could determined the red shift
in wavelength using the nanodrop technique after completing the synthesis procedure.

# Test:

![](https://static.igem.wiki/teams/4636/wiki/engineering-success/figure-10.png)

Figure 10. The comparison of wavelengths between the pure gold nanoparticle solution and solution after processing Option 2.

Fortunately, we had found out
another protocol, named it Option 2 (see page experiment for protocols) [[11]](#[11]), for conjugating
probes and gold nanoparticles. As mentioned in the cycle one, we could determined the red shift
in wavelength using the nanodrop technique after completing the synthesis procedure.

# Learn:

According to Figure 10, they show
significant red shift in wavelength. In the other word, we successfully attached probes on the
surface of gold nanoparticles.

# Section 4: Model

# Cycle : Difussion

# Purpose:

According to scientific research,
we
know that the segregation of the gold nanoparticles may influence the observed color of the
solution. Therefore, in order to predict the color of the solution, we need to investigate the
diffusion process of the gold nanoparticles beforehand.

# Design:

We use the function and the
geometric
factors of the container to simulate the diffusion of the particles. After getting the
equilibrium
condition of the diffusion result, we then try to comprehend the distribution of every particle
to
further derive the wavelength shift of the liquid.

# Redesign:

Initially, we assume the velocity inlet to be constant, the result is show in Figure 11:

![](https://static.igem.wiki/teams/4636/wiki/engineering-success/engineeringfig11.png)

Figure 11. Diffusion under constant velocity

We then use the Gaussian
distribution
to fit with Figure 11 to see the distribution extent. At first, we use the variances of 1, 5 and
20
then choose the most similar case to fit with Figure11 The results are given as follows:

# Build:

However, the constant descending
velocity does not fit the actual condition since the velocity may decrease along the descending
process. Then we change descending velocity into a time-depending function to more explictly
simulate the real condition. The diffusion result can be shown as:

# Test:

![](https://static.igem.wiki/teams/4636/wiki/engineering-success/engineeringfig12.png)

Figure 12. Gaussian distribution of variance 1, 5 and 20

However, even with the most familiar in
those three cases, there is still a significant difference between the simulation and the result. In
order to retrieve the most familiar variance, we use the following code to compare the pixel between
distribution under different variances and Figure 11 Hence, the variance can be elaborated as follows:

![](https://static.igem.wiki/teams/4636/wiki/engineering-success/engineeringfig13.png)

Figure 13. Difference of pictures under different variances

Worth mentioning, when the variance is
greater than 1 then the segregation area will be much smaller than Figure 1. Therefore, we choose the
lowest point in the graph to do the distance calculation. The comparison between the most familiar
variance and Figure 1 are shown as follows:

![](https://static.igem.wiki/teams/4636/wiki/engineering-success/engineeringfig14.png)

Figure 14. Comparison between the most familiar variance and Figure 11

Moreover, we also retrieve the value directly from the Simscale:

![](https://static.igem.wiki/teams/4636/wiki/engineering-success/engineeringfig15.png)

Figure 15. Turbulent kinetic energy of every particle

In order to calculate the distance
between particles, we assume that the particle in the container is homogenous and distribute
uniformly, therefore the kinetic energy is proportional to the number of particles. The result
is given as:

| Volume (m3) | Moles (mol) | Max distance (m) | Min distance (m) |
| --- | --- | --- | --- |
| 7.5×10-8 | 9.75×10-13 | 5.04×10-7 | 5.03×10-7 |

Sheet 1. Calculation of distance between particles in every region

# Build:

However, the constant descending
velocity does not fit the actual condition since the velocity may decrease along the descending
process. Then we change descending velocity into a time-depending function to more explictly
simulate the real condition. The diffusion result can be shown as:

![](https://static.igem.wiki/teams/4636/wiki/engineering-success/engineeringfig16.png)

Figure 16. Diffusion under time-depending velocity

# Learn:

After determinating that diffusion are
mainly cause by the velocity inlet and the osmotic pressure which caused by the difference of the
concentration, we know can further derive the relationship as follows:

π
=
i
c
R
T

F
/
A
=
i
c
R
T

1
A
×

m
t
v
=
i
c
R
T

v
=

A
i
R
T
c
t
/
m

v
(t)
=
K
⋅
t
/
M
+
constant

Figure 17. Derivation of the velocity relationship

# Redesign:

After knowing the velocity
relationship, we apply it back to the simulation to understand the influence of different
concentration. The simulation results are given in the model page.

# Section 5: Model (RT-RPA)

# Cycle : Amplification

# Purpose:

The establishment of RT-RPA model
is to assist Wetlab in determining whether the current experimental design can yield the
expected results through simulation before the actual experiment.

# Design:

We first select the crucial reactions of RT-RPA and converted these reactions into differential equations. Then, we use SimBiology to execute these differential equations.

# Build:

At the beginning, we intended to
directly utilize the differential equations from the paper and incorporate them with the
differential equations for reverse transcriptase. However, after running simulations, we found
that we were unable to obtain results consistent with the paper.

![](https://static.igem.wiki/teams/4636/wiki/engineering-rtrpa/engineering-1.jpg)

Figure 18. Simulation Results of cDNA after a 20 minutes RPA Reaction

# Learn:

After referring to the modeling approach of previous iGEM teams, we decided to independently select the crucial reactions of RT-RPA and input them into SimBiology for simulating these reactions.

# Redesign:

After determining the new approach to constructing the model, we referred to the relevant paper and selected the crucial reactions of RT-RPA. We then inputted these reactions into SimBiology to establish the model.

G + F ⇌ FG (1)

FG + R ⇌ FGR' (2a)

FGR' → FGRi + G (2b)

FGRi + R ⇌ FGRc (2c)

FGRc → FRc + G (2d)

FRc + D ⇌ FRcD (3)

FRcD + ATP → FD + R + AMP + PPi (4a)

FRcD + ATP → FD + ADP + H3PO4 (4b)

FD + P ⇌ PFD (5)

PFD + dNTPs → P + PPi + D (6)

This is what the complete RT-RPA
model look like in simbiology:

![](https://static.igem.wiki/teams/4636/wiki/engineering-rtrpa/engineering-2.png)

Figure 19. RT-RPA reaction pathway in MATLAB Simbiology

After identifying those crucial
reactions of RT-RPA, we referred to relevant paper to obtain the required parameters and initial
values. In addition, we also obtained initial values for some key reactants from Wetlab.
Ultimately, we successfully get the simulation results we expected.

![](https://static.igem.wiki/teams/4636/wiki/engineering-rtrpa/engineering-3.jpg)

Figure 20. Simulation Results of cDNA after a 20 minutes RPA Reaction

After obtaining the simulated
results we expected, we aim to apply the model in practical scenarios. We decided to use this
RT-RPA model to validate Wetlab's proposition that the quantity of the forward primer influences
the lower limit of the initial values of cDNA. The simulation results showed that the primer
quantity does influence the lower limit of the initial value of cDNA. This not only confirmed
our proposition but also boosted our confidence in the RT-RPA model.

![](https://static.igem.wiki/teams/4636/wiki/engineering-rtrpa/engineering-4.jpg)

Figure 21. simulation results of cDNA after a 40 minutes RPA reaction when the forward primer quantity is 4.8e-7

![](https://static.igem.wiki/teams/4636/wiki/engineering-rtrpa/engineering-5.jpg)

Figure 22. simulation results of cDNA after a 40 minutes RPA reaction when the forward primer quantity is 4.8e-8

# Reference

[1] Chen, H., Cheng, K., Liu, X., An, R.,
Komiyama, M., & Liang, X. (2020). Preferential production of RNA rings by T4 RNA ligase 2
without any splint through rational design of precursor strand. *Nucleic acids research*,
**48**(9), e54. <https://doi.org/10.1093/nar/gkaa181>

[2] An, R., Li, Q., Fan, Y., Li, J., Pan,
X., Komiyama, M., & Liang, X. (2017). Highly efficient preparation of single-stranded DNA rings
by T4 ligase at abnormally low Mg(II) concentration. *Nucleic acids research*,
**45**(15), e139. <https://doi.org/10.1093/nar/gkx553>

[3] Jinek, M., Coyle, S. M., & Doudna, J.
A. (2011). Coupled 5' nucleotide recognition and processivity in Xrn1-mediated mRNA decay.
*Molecular cell*, **41**(5), 600–608. <https://doi.org/10.1016/j.molcel.2011.02.004>

[4] Boss, M., & Arenz, C. (2020). A Fast
and Easy Method for Specific Detection of Circular RNA by Rolling-Circle Amplification.
*Chembiochem : a European journal of chemical biology*, **21**(6), 793–796. <https://doi.org/10.1002/cbic.201900514>

[5] Cui, Y., Han, X., An, R., Zhou, G.,
Komiyama, M., & Liang, X. (2018). Cyclization of secondarily structured oligonucleotides to
single-stranded rings by using *Taq* DNA ligase at high temperatures. *RSC advances*,
**8**(34), 18972–18979. <https://doi.org/10.1039/c8ra02804d>

[6] <http://rna.tbi.univie.ac.at//cgi-bin/RNAWebSuite/RNAfold.cgi?PAGE=3&ID=zcRAvdofnB&r=65>

[7] Boss, M., & Arenz, C. (2020). A Fast
and Easy Method for Specific Detection of Circular RNA by Rolling-Circle Amplification.
*Chembiochem : a European journal of chemical biology*, **21**(6), 793–796. <https://doi.org/10.1002/cbic.201900514>

[8] Maguire, S., & Guan, S. (2022). Rolling
circle reverse transcription enables high fidelity nanopore sequencing of small RNA. *PloS
one*, **17**(10), e0275471. <https://doi.org/10.1371/journal.pone.0275471>

[9] Ali, M. M., Kanda, P., Aguirre, S. D.,
& Li, Y. (2011). Modulation of DNA-modified gold-nanoparticle stability in salt with
concatemeric single-stranded DNAs for colorimetric bioassay development. *Chemistry (Weinheim
an der Bergstrasse, Germany)*, **17**(7), 2052-2056. <https://doi.org/10.1002/chem.201002677>

[10] Li, F., Zhang, H., Dever, B., Li, X.
F., & Le, X. C. (2013). Thermal stability of DNA functionalized gold nanoparticles.
*Bioconjugate chemistry*, **24**(11), 1790-1797. <https://doi.org/10.1021/bc300687z>

[11] Liu, J., & Lu, Y. (2006). Preparation
of aptamer-linked gold nanoparticle purple aggregates for colorimetric sensing of analytes.
*Nature protocols*, **1**(1), 246-252. <https://doi.org/10.1038/nprot.2006.38>

[12] R. Tsekov, P. Georgiev, S. Simeonova and K. Balashev (2017), Quantifying the Blue Shift in the Light Absorption of Small Gold Nanoparticles <https://arxiv.org/ftp/arxiv/papers/1702/1702.04513.pdf>

[13] Perez-Juste J, Liz- Marzán LM, Carnie S, Chan DYC, Mulvaney P (2004) Electric-field-directed growth of gold nanorods in aqueous surfactant solutions <https://doi.org/10.1002/adfm.200305068>

[14] Morris AM, Watzky MA, Finke RG (2009) Protein aggregation kinetics, mechanism, and curve-fitting: a review of the literature <10.1016/j.bbapap.2008.10.016>

[15] Lewis GN (1908). The Osmotic Pressure of Concentrated Solutions and the Laws of the Perfect Solution. Journal of the American Chemical Society. <https://pubs.acs.org/doi/abs/10.1021/ja01947a002>

[16] Liu, Jie ; Berger, Christopher L ; Morrical, Scott W (2013). Kinetics of Presynaptic Filament Assembly in the Presence of Single-Stranded DNA Binding Protein and Recombination Mediator Protein. Biochemistry (Easton), 2013, Vol.52 (45), p.7878-7889 <https://pubs.acs.org/doi/full/10.1021/bi401060p>

[17] Moody, Clint ; Newell, Heather ; Viljoen, Hendrik (2016). A mathematical model of recombinase polymerase amplification under continuously stirred conditions. Biochemical engineering journal, 2016, Vol.112, p.193-201 <https://www.sciencedirect.com/science/article/pii/S1369703X16301152?casa_token=nv3pO9UHxvYAAAAA:9kA9Wc27RzRSbEKsF5uC_q44f_6Z-g4MGT-T63K7gns8uvCOv2T9ttnD-UmuxCmZKDGIWak_roM#bib0090>

[18] Farb, Joshua N. ; Morrical, Scott W. (2009). Role of Allosteric Switch Residue Histidine 195 in Maintaining Active-Site Asymmetry in Presynaptic Filaments of Bacteriophage T4 UvsX Recombinase. Journal of molecular biology, 2009, Vol.385 (2), p.393-404 <https://www.sciencedirect.com/science/article/pii/S0022283608014125>

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block page\_content %}

Experiments

* [Sequence Design](#Sequence Design)
* [Cloning](#Cloning)

+ [Ligation](#Ligation)
+ [Transformation](#Transformation)
+ [Colony PCR](#Colony PCR)
+ [Plasmid extraction](#Plasmid extraction)

* [All-aspect](#PCR)

+ [Plasmid PCR](#Plasmid PCR)
+ [Insert PCR](#Insert PCR)

* [In Vitro Transcription
  (IVT)](#In Vitro Transcription (IVT))
* [Circularization](#Circularization)
* [CircRNA enrichment](#CircRNA enrichment)
* [AuNPs synthesis](#AuNPs synthesis)
* [Probe conjugated AuNPs](#Probe conjugated AuNPs)

+ [[Option 1] Probe
  conjugated-AuNP synthesis](#[Option 1] Probe conjugated-AuNP synthesis)
+ [[Option 2] Probe
  conjugated-AuNP synthesis](#[Option 2] Probe conjugated-AuNP synthesis)
+ [X-ray photoelectron
  spectroscopy (XPS) Analysis](#X-ray photoelectron spectroscopy (XPS) Analysis)

* [RCA](#RCA)

+ [Using ProtoScript® II
  Reverse Transcriptase](#Using ProtoScript® II Reverse Transcriptase)
+ [Using
  Induro® Reverse Transcriptase](#Using)

* [Probe conjugated-AuNP
  detection](#Probe conjugated-AuNP detection)
* [Repeated insert
  sequence synthesis](#Repeated insert sequence synthesis)

+ [Insert
  digestion](#Insert digestion)
+ [Insert ligation](#Insert ligation)

* [Preparation of
  template for AELA-PCR](#Preparation of template for AELA-PCR)

+ [Ligation
  with vector](#Ligation with vector)
+ [Transformation](#Transformation)
+ [Colony PCR](#Colony PCR)
+ [Plasmid extraction](#Plasmid extraction)

* [AELA-PCR](#AELA-PCR)
* [RPA](#RPA)
* [RT-RPA](#RT-RPA)
* [PCRD](#PCRD)
* [Reference](#Reference)

# Sequence Design

All necessary DNA sequences for
each step must be designed before beginning the experiment. Please refer to the [Parts](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27parts%27%29%20%7D%7D) for more
details.

# Cloning

# Ligation

# Equipment

1. Dry bath

# Consumables

1. 2X Reaction Buffer (Thermo Fisher Scientific)
2. pJET1.2/blunt Cloning Vector (50 ng/µL) (Thermo Fisher Scientific)
3. T4 DNA Ligase (400,000 U/mL) (New England Biolabs)
4. Sample (insert)
5. Nuclease-free water (Protech Technology)

# Protocol

1. Set up the ligation reaction on ice. Vortex briefly and centrifuge for 3-5 s.

2. Incubate the ligation mixture at room temperature (22 °C) for 30 min.
3. Use the ligation mixture directly for transformation.

# Transformation

# Equipment

1. Dry bath
2. Disposable cell spreaders
3. Incubator

# Consumables

1. DH5-alpha E. coli (New England Biolabs)
2. Sample (ligation product)
3. LB medium plate with ampicillin
4. LB medium with ampicillin (0.1 mg ampicillin / 1 mL LB)

# Protocol

1. Pipette 100 µL of DH5-alpha E. coli from the stock and then aliquot 50 µL into another
eppendorf tube. Place the tube on ice.

2. Add 5 µL of ligation product to the aliquoted DH5-alpha E. coli, flick the tube, and
incubate it on ice for 25 minutes, flicking gently every ten minutes.

3. Place both tubes in a 42°C dry bath for 45 seconds.

4. In a laminar flow hood, add 1 ml of LB medium with ampicillin to each tube, and incubate
them
at 37°C with shaking for 1 hour.

5. Take 100 µL of the mixture and spread it evenly on a LB medium plate with ampicillin
using
a disposable cell spreaders.

6. Incubate the LB medium plate with ampicillin in an incubator at 37°C for 16 hours.

# Colony PCR

# Equipment

1. Thermocycler
2. Culture tube
3. Dry bath
4. Incubator

# Consumables

1. LB medium with ampicillin (0.1 mg ampicillin / 1 mL LB)
2. 2X Master Mix (Protech Technology)
3. pJET1.2 Forward Sequencing Primer, 10 µM (Thermo Fischer Scientific)
4. pJET1.2 Reverse Sequencing Primer, 10 µM (Thermo Fischer Scientific)
5. Sample (transformation product)
6. Nuclease-free water (Protech Technology)

# Protocol

1. Use pipette tips to pick colony, each petri dish choose 4 colonies.

2. Put the colony in 200 µL of LB with ampicillin (0.1 mg ampicillin / 1 mL LB).

3. Shake in a 37°C incubator for 2 hours in preparation for colony PCR.

4. Add the following reagents to PCR tube.

5. React in a thermocycler using following setting.(x 35 cycles)

6. Perform 1.5% agarose gel electrophoresis.

7. Identify samples with the correct sequence length and no unwanted bands. Then transfer 10
µL
succefully transformed bacteria liquid to 5 mL LB with ampicillin in culture tube.

8. Shake in a 37°C incubator for 16 hours.

# Plasmid extraction

# Equipment

1. Dry bath
2. Centrifuge

# Consumables

1. 50% Glycerol
2. Mini Plus Plasmid DNA Extraction System (Viogene)
3. Sample (colony PCR product)

# Protocol

1. Take 500 µL of a 16-hour bacterial culture and use it for bacterial cryopreservation. Mix the
500 µL bacterial culture with 500 µL of glycerol in an Eppendorf tube, then store it at -80
°C.
2. Take 1 mL of the remaining 4.5 mL bacterial culture at a time and centrifuge it 1 min at
10,000rpm in Eppendorf tubes. After each centrifugation, discard the supernatant using a
pipette.
3. Add 200 µL Solution1 and pipette it until the cells are completely resuspended.
4. Add 200 µL Solution2 and mix by gently inverting the capped tube 5-6 times. Incubate at
room temperature for 5 min.
5. Add 300 µL Solution3 and mix by gently inverting the capped tube 5-6 times. Incubate at room
temperature for 3 min.
6. Centrifuge it at 10,000 rpm for 5 minutes. You will observe the formation of a dense, white
pellet either along the side or at the base of the tube.
7. Load the spin column into a collection tube, transfer the supernatant from step 6 directly to
spin column, spin for 1 min at 10,000 rpm.
8. Take out the spin column from the collection tube, dispose of the liquid that flows through
it, reinsert the spin column, and then apply 700 µL of washing solution. Centrifuge for 1
minute.
9. Take out the spin column from the collection tube, pour away the liquid, reinsert the spin
column, and then apply 700 µL of washing solution. Centrifuge for 1 minute.
10. Dispose of the filtrate and then centrifuge at 10,000 rpm for 3 minutes to eliminate any
remaining traces of ethanol. Incubate the spin column in a 60 °C oven for 8 minutes.
11. Remove the spin column and place the column in a new Eppendorf tube.
12. Add 50 µL free water into the column. Elute DNA by centrifugation for 1 min and store the
eluted DNA at -20 °C.

# All-aspect PCR

# Plasmid PCR

# Equipment

1. PCR tubes
2. Thermocycler

# Consumables

1. 2X Master Mix (Protech Technology)
2. Nuclease-free water (Protech Technology)
3. Forward primer (10 μM)
4. Reverse primer (10 μM)
5. Sample (plasmid)
6. Gel/PCR DNA Isolation System (Viogene)

# Protocol

1. Add the following reagents to PCR tube.

|  |  |
| --- | --- |
| Reagents | Amount |
| 2X Master Mix | 10 μL |
| Foward primer | 0.5 μL |
| Reverse primer | 0.5 μL |
| Sample | 2 μL |
| Nuclease-free water | to 20 μL |
| Total | 20 μL |

2. React in a thermocycler at following setting.

|  |  |  |  |
| --- | --- | --- | --- |
| Stage | Temperature | Time |  |
| Denaturation | 95 °C | 5 min |  |
| Denaturation | 95 °C | 30 sec | 35 cycles |
| Annealing | 61 °C | 30 sec |
| Extension | 72 °C | 1 min |
| Extension | 72 °C | 7 min |  |
| Cooling | 4 °C | ∞ |  |

3. Run the plasmid PCR DNA on a 1.5 % agarose gel.
4. Conduct gel purification using Gel/PCR DNA Isolation System followed the provided
protocol.
5. Measure a DNA concentration of the purified product by NanoDrop.

# Insert PCR

# Equipment

1. PCR tubes
2. Thermocycler

# Consumables

1. Pfu Buffer (5 U/µL) (Protech Technology)
2. Pfu Polymerase (5 U/µL) (Protech Technology)
3. Forward primer (10 μM)
4. Reverse primer (10 μM)
5. dNTP (10 mM) (Thermo Fischer Scientific)
6. Sample (template)
7. Gel/PCR DNA Isolation System (VIOGENE)

# Protocol

1. Add the following reagents to PCR tube.

|  |  |
| --- | --- |
| Reagents | Amount |
| Pfu Buffer | 5 μL |
| Foward primer | 1 μL |
| Reverse primer | 1 μL |
| dNTP | 1 μL |
| Pfu pol | 0.25 μL |
| Template (plasmid) | 1 μL |
| Nuclease-free water | to 50 μL |
| Total | 50 μL |

2. React in a thermocycler at following setting.

|  |  |  |  |
| --- | --- | --- | --- |
| Stage | Temperature | Time |  |
| Denaturation | 95 °C | 5 min |  |
| Denaturation | 95 °C | 1 min | 35 cycles |
| Annealing | 61 °C | 30 sec |
| Extension | 68 °C | 1 min |
| Extension | 68 °C | 10 min |  |
| Cooling | 4 °C | ∞ |  |

3. Run the insert PCR DNA on a 1.5 % agarose gel.
4. Conduct gel purification using Gel/PCR DNA Isolation System followed the provided
protocol.
5. Measure a DNA concentration of the purified product by NanoDrop.

# In Vitro Transcription (IVT)

# Equipment

1. Dry bath
2. NanoDrop Spectrophotometers

# Consumables

1. Nuclease-free water (Protech Technology)
2. 10X RNA Pol Reaction Buffer (New England Biolabs)
3. NTP (100 mM) (Thermo Fisher Scientific)
4. RNase Inhibitor (40,000 U/mL) (New England Biolabs)
5. DTT (0.1 M) (Thermo Fisher Scientific)
6. T7 RNA Polymerase (50,000 U/µL) (New England Biolabs)
7. GeneJET RNA Cleanup and Concentration Micro Kit (Thermo Fischer Scientific)

# Protocol

1. Mix the following reagents at room temperature. The T7 RNA polymerase should be added last.

|  |  |
| --- | --- |
| Reagents | Amount |
| 10X RNA Pol Reaction Buffer | 2 μL |
| NTP | 0.1 μL for each |
| RNase Inhibitor | 1 μL |
| DTT | 1 μL |
| cDNA (1 μg) | Variable |
| T7 RNA poly | 2 μL |
| Nuclease-free water | to 20 μL |
| Total | 20 μL |

2. Incubate at 37 °C for 1 hr.
3. In order to eliminate DNA template used for in vitro transcription (IVT) process, we added DNase
I for DNA degradation.

DNase I calculation method:
a. According to the product information page of Thermo
Fisher[[1]](#[1]) two units of DNase I are required to degrade 1 μg of DNA. As a
reference, we could
calculate the amount of DNA to be digested in order to determine the required amount of DNase I
to be added.
b. In the process of in vitro transcription (IVT), we utilize
a fixed amount of DNA template. The weight of the DNA we used is 500 ng. 500 ng = 0.5 μg of DNA;
therefore, the required DNase I is 1 unit. Since the concentration of DNase I is 1 U/μL, the
amount of DNase I needed in this process is 1 μL.

4. After adding DNase I, incubate the mixture at 37 °C for 15 minutes.
5. Follow the DNase I clean-up protocol of GeneJET RNA Cleanup and Concentration Micro Kit
(Thermo Fischer Scientific)
6. Use NanoDrop to measure the concentration of sample.

# Circularization

# Equipment

1. PCR tubes
2. Thermocycler
3. NanoDrop Spectrophotometers

# Consumables

1. Sample (IVT product)
2. 18 bp Splint (25 µM)
3. 16 bp Splint (25 µM)
4. 14 bp Splint (25 µM)
5. Pyrophosphatase (1 U/µL)(Thermo Fischer Scientific)
6. T4 RNA ligase 2 (New England Biolabs)
7. T4 RNA ligase 2 Reaction Buffer (New England Biolabs)
8. DNase I (1 U/µL) (Thermo Fischer Scientific)
9. GeneJET Plasmid Miniprep Kit (Thermo Fischer Scientific)
10. RNase-free water (Protech Technology)

# Protocol

**Monophosphorylation**

1. In order to monophosphorylate the RNA sample from above-mentioned process, pyrophosphatase is
the enzyme required. According to product page of Thermo Fisher[[1]](#[1]), one unit of
pyrophosphatase
enzyme could catalyzes 1 μmol of inorganic pyrophosphate. As a reference, we could calculate the
mole of RNA to be monophosphorylated, in order to determine the required amount of
pyrophosphatase to be added.
2. The total amount of product could be calculated by following formulas:

a. Mass of RNA sample: Concentration of RNA × Total volume required to be monophosphorylated = Mass
of RNA sample
b. Mole of RNA sample: (Mass of RNA sample) / (Molecular weight of sequence) = Mole of RNA
sample
c. Pyrophosphatase to be added: Mole of RNA sample (the unit should be convert to μmol) = Unit of
pyrophosphatase enzyme to be added

3. Incubate the RNA sample that has added monophosphatase at 25°C, for 30 min.
4. Verification of Monophosphorylation:

a. In order to verify the success of monophosphorylation, we utilize an enzyme, XRN-1. XRN-1 is
an exoribonuclease enzyme that could degrade RNA sequences with a 5' monophosphate group.
Therefore, RNA that is successfully monophosphorylated could be disintegrated by XRN-1.
b. Add the following components for verification:
The sample required is 1 μg, so the volume of sample to be added could be calculate as 1
μg/concentration of monophosphorylated sample.
c. Incubate for 60 minutes at 37 °C.

|  |  |
| --- | --- |
| Reagents | Amount |
| XRN-1 | 1 μL |
| 10X reaction buffer | 1 μL |
| Sample | 1 μg |
| RNase-free water | to 10 μL |
| Total | 10 μL |

5. Follow the protocal of GeneJET Plasmid Miniprep Kit (Thermo Fischer Scientific).
6. Use NanoDrop to measure the concentration of sample.

**Formation of dsRNA**

1. We utilize T4 RNA ligase 2 to back-splice the linear RNA, which requires forming dsRNA using
ssRNA combined with splint.
2. Splint should be added, and the volume could be calculated by following formulas:

a. Calculate the mole of our ssRNA product: Concentration of RNA × Volume of ssRNA product ÷
Molecular weight of the sequence = Mole of ssRNA product (the unit should be convert to pmol)
b. Calculate the mole of splint should be added: Since the molar ratio of splint : ssRNA
should be 2 : 1, the mole of splint = mole of ssRNA × 2.
c. Calculate the volume of splint should be added: The mole of splint ÷ Concentration of the
splint = Volume of splint

3. Incubate the mixture at 95°C for 2 min and turn off thermocycler to let it cool down for 1.5 hr.

**Back splicing**

1. Calculate the total volume of dsRNA product from last step: Volume of splint + Volume of ssRNA =
Volume of dsRNA product.
2. Calculate the concentration of dsRNA mixture: Mole of ssRNA product ÷ Volume of dsRNA product =
Concentration of dsRNA mixture
3. Calculate the volume of dsRNA product required for circularization: The ideal mole of
circularization reagent is 20 µmol. So we could calculate the required volume of dsRNA product by 20
µmol ÷ Concentration of dsRNA mixture.
4. Add the components as following chart:

|  |  |
| --- | --- |
| Reagents | Amount |
| 10X T4 RNA ligase 2 Reaction Buffer | 2 μL |
| T4 RNA ligase 2 | 1 μL |
| dsRNA product | Variable |
|
 RNase-free water | to 20 μL || Total | 20 μL |

5. Incubate the mixture at 25°C for 1hr.
6. In order to eliminate splint DNA used in circularization process, we add DNase I for DNA
degradation.

a. Two units of DNase I is required to degrade 1 μg of DNA. As a reference, we could calculate
the amount of splint DNA to be digested, in order to determine the required amount of DNase I to
be added.
b. Calculate the mass of the added splint DNA : In original tube of splint, it indicates the
volume and mass of splint. By these two value, we could calculate the mass of splint (µg) per
volume (µL). Then the volume of added splint DNA could be calculate by mass (µg) of splint per
volume (µL) × Volume of splint added = Mass of added splint DNA (µg)
c. Calculate the DNase I should be added: Mass of added splint DNA (μg) × 2 = Unit of DNase
should be added.

7. Follow the DNase I clean-up protocol of GeneJET RNA Cleanup and Concentration Micro Kit
(Thermo Fischer Scientific).

# CircRNA enrichment

# Equipment

1. Dry bath
2. E-Gel™ Power Snap Electrophoresis System (Invitrogen™)

# Consumables

1. RNase R (10 U/μl) (abcam)
2. RNase inhibitor (40,000 U/ml) (New England Biolabs)
3. 10X RNase R Reaction Buffer (abcam)
4. Nuclease-free water (Protech Technology)
5. Low range RNA ladder or Century™-Plus RNA Markers (Thermo Fischer Scientific)
6. 2X RNA loading dye (New England Biolabs)

# Protocol

**RNase R degradation**

1. RNase R - Eliminate linear form RNA:

After the circularization process, we are supposed to
transform all the linear RNA into circRNA. However, research related to circularization has not
yet achieved a 100% circularization rate. Therefore, in order to obtain pure circRNA, we use
RNase R to eliminate failed circularization products, which are linear RNAs, to get a higher
percentage of circRNA for further experiments.

2. Calculate RNase R required for degrading linear RNA:

a. According to product information page of Abcam[[3]](#[3]), one unit of RNase R could
convert 1 µg of
poly(A) into nucleotides. As a reference, we should calculate the total mass of RNA for further
degradation.
b. Mass of total RNA sample: Concentration of RNA (calculated by previous step by Nanodrop)
× Total volume of RNA = Mass of total RNA sample.
c. Unit of RNase R to be added: Mass of total RNA sample (the unit should be convert to μg) =
Unit of RNase R to be added
d. Volume of RNase R to be added: According to Abcam website[4],
concentration of RNase R is 10 U/µL, so the volume of RNase R to be added = Unit of RNase R÷10.

3. Add the following components:

|  |  |
| --- | --- |
| Reagents | Amount |
| RNA sample | 1 μL to 10 μL |
| RNase R | 0.2 μL to 2 μL (according to RNA sample) |
| RNase Inhibitor | 0.5 μL |
| 10X RNase R Reaction Buffer/td> 2 μL | |
| Nuclease-free water | to 20 μL |
| Total | 20 μL |

4. Incubate the mixture at 37°C for 2hr.
5. Follow protocol of GeneJET Plasmid Miniprep Kit (Thermo Fischer Scientific).

**E-gel EX electrophoresis**

Sample preparation

1. Prepare the sample and mix it with dye in the ratio of one to one.
2. There are ten wells in one E-Gel EX cassette. If the number of loading samples is less than ten,
the empty wells should be filled with water and dye in a one-to-one ratio.
3. The RNA ladder with dye and samples with dye should all be denatured before loading them into
E-Gel EX cassette wells. For RNA ladder denaturation, it has to be denatured at 90 °C for 2 minutes.
While for the denature of the sample, it has to go under 65 °C for 15 minutes.

Operation

1. Open up the Thermo Fisher Scientific E-Gel EX package.
2. Take out the E-Gel EX cassette and place it into the E-Gel Power Snap System. Make sure the
electrode is placed correctly.
3. Make sure that the lid of the E-Gel EX cassette has been removed before loading the sample.
4. After the sample has all been loaded, close the lid of the E-Gel Power Snap System.
5. Turn on the E-Gel Power Snap System and set the options and parameters according to your
experiment.
6. After the setting is done, press start to run gel.
7. The results can be observed under the E-Gel Power Snap System hood simultaneously during the run.

# AuNPs synthesis

# Equipment

1. Two-neck flask
2. Liebig condenser
3. Heating mantle
4. Hot plate/magnetic stirrer
5. Thermometer
6. Automatic pipette
7. Stir Bar
8. 50 mL centrifuge tubes

# Consumables

1. HAuCl4 powder
2. Deionized water
3. 1% Sodium citrate
4. Aluminum foil

# Protocol

1. 3.1 mM HAuCl4 5.69 mL + 14.11 mL deionized water mixed in two-neck flask, put a
magnetic
stir bar in the two-neck flask.
2. Heat it in a heating mantle until boiling (130 °C, ocean sand, approximately 10 min), while
gently stirring it (60 rpm). **\*Note: Put the condenser at one of the neck, the other neck closed
by a stopper (Aluminum
foil).**
3. Turn the stir to 400 rpm.
4. Add 2.4 mL 1% sodium citrate in the two-neck flask (remember to add it all at once!).
5. When the color changes into burgundy red, keep heating for 10-15 min.
6. Remove the heating mantle, continue stirring.
7. After cooling down, pour all into a centrifugation tube.
8. Test it with NanoDrop spectrophotometer, its peak wavelength will be close to 520 nm.

# SEM/EDS Analysis

**Equipment**

1. Diamond glass cutter
2. 250 mL beaker
3. Ultrasonic cleaner
4. Laboratory oven
5. Vacuum dryer
6. SEM-IT800
7. IT800-EDS

**Consumables**

1. Silicon wafer
2. Dish detergent
3. Ethanol
4. Acetone
5. Petri dish

**Protocol**

Sample Preparation

1. Cut the silicon wafer to approximately 0.4 cm\*0.4 cm square with a diamond glass cutter.
2. Put the silicon wafer into a clean beaker. Wash the silicon wafer with diluted dish detergent
in an ultrasonic cleaner for 12 minutes.
3. Transfer the silicon wafer to another clean beaker. Wash the silicon wafer with ddH2O in an
ultrasonic cleaner for 12 minutes. Repeat this step three times.
4. Transfer the silicon wafer to another clean beaker. Wash the silicon wafer with ethanol in an
ultrasonic cleaner for 12 minutes.
5. Transfer the silicon wafer to another clean beaker. Wash the silicon wafer with acetone in an
ultrasonic cleaner for 12 minutes.
6. Transfer the silicon wafer to another clean beaker. Wash the silicon wafer with ddH2O in an
ultrasonic cleaner for 12 minutes.
7. Transfer it to a clean petri dish. Dry it in an oven.
8. Drop 1 µL of nanoparticles sample on a silicon wafer.
9. Put it in a vacuum dryer for 1-2 hours, confirm the sample is completely dry.

Analysis

1. Using SEM-IT800 to observe the shape of gold nanoparticles (AuNPs) and measure their
diameter. Additionally, utilizing IT800-EDS to analyze the elemental composition and
distribution in the sample.

# Probe conjugated AuNPs

# [Option 1] Probe conjugated-AuNP synthesis

# Equipment

1. Disposable scintillation vials
2. Centrifuge
3. 1.5 mL microcentrifuge tube

# Consumables

1. Gold nanoparticles (3 mL, 36 pmol, O.D. = 3.24)
2. Deionized water
3. 1M Tris-HCl (pH 7.5)
4. 1 M NaCl
5. 10 mM TCEP
6. DNA probes (1 mM)
7. ddH2O
8. 500 mM acetate buffer (pH 5.2)
9. 1M NaCl
10. Binding buffer (3 mL, 20 mM Tris-HCl, pH 7.5, 100 mM NaCl)

# Protocol

1. Soak two disposable scintillation vials (20 mL volume) in 12 M NaOH for 1 hr at room
temperature. Rinse the vials with copious amounts of deionized water.
2. Prepare 10 mM TCEP
3. Pipette 1.8 µL of 1 mM DNA, 7.2 μL ddH2O into a 1.5 mL microcentrifuge tube.
4. Add 1 μL of 500 mM acetate buffer (pH 5.2) and 1.5 µL of 10 mM TCEP to each tube to activate
the thiol-modified DNA. Incubate the sample at room temperature for 1 hr.
5. Gold nanoparticles (3 mL, O.D.= 3.24) was incubated at room temperature with the thiol-modified
DNA oligonucleotide (1800 pmol) for 24 hr.
6. Tris-HCl (30 μL, 1 M, pH 7.5; all pH values were measured at 23 °C) and an aqueous solution
of NaCl (270 μL, 1 M) were added, mixed, and incubated at RT for another 24 h.
7. Tris-HCl (15 μL, 1M, pH 7.5) and aqueous solution of NaCl (50 μL, 1M) were added and further
incubated for 24 h.
8. The aged AuNPs were isolated by centrifugation at 15,000 g at RT for 10~30 mins, re-suspended
in the binding buffer, and stored at RT.

# [Option 2] Probe conjugated-AuNP synthesis

# Equipment

1. Disposable scintillation vials
2. Centrifuge
3. 1.5 mL microcentrifuge tube

# Consumables

1. 1 mM DNA
2. TCEP (10mM)
3. 12 M NaOH
4. 500 mM acetate buffer (pH 5.2)
5. Gold nanoparticles (O.D.=3.5)
6. Nuclease-free water (Protech Technology)
7. 500 mM Tris acetate (pH 8.2) buffer
8. 1 M NaCl
9. Binding buffer (20 mm Tris-HCl, pH 7.5, 100 mm NaCl)

# Protocol

1. Soak two disposable scintillation vials (20 mL volume) in 12 M NaOH for 1 hr at room
temperature. Rinse the vials with a copious amount of deionized water.
2. Prepare 10 mM TCEP
3. Pipette 9 µL of 1 mM DNA probe into a 1.5 mL microcentrifuge tube.
4. Add 1 µL of 500 mM acetate buffer (pH 5.2) and 1.5 µL of 10 mM TCEP to each tube to activate
the thiol-modified DNA. Incubate the sample at room temperature for 1 hr.
5. Transfer 3 mL of the already prepared gold nanoparticles (O.D. value 3.5) to each of the two
NaOH-treated glass vials, and then add the TCEP-treated thiol DNA with gentle shaking by hand
(or pipetting), and then mix the solution by stir bar for 16 hr (avoid light).
6. Add 30 µL of 500 mM Tris acetate (pH 8.2) buffer dropwise to each vial with gentle hand
shaking. The final Tris acetate concentration is 5 mM.
7. Add 300 µL of 1 M NaCl dropwise to each vial with gentle hand shaking. Store the two vials in
a drawer for at least another day before use (avoid light).
8. Centrifuge at 10,000 rpm, 20 mins. (The centrifugation condition might change.) **\*Note: Adjust the centrifugation condition (duration and/or speed): The supernatant after
centrifugation should be as transparent as possible, instead of a pink color. Also, there
shouldn't be any black dots remaining on the eppendorf's wall, which indicates there's
unfavorable aggregation of gold nanoparticles.**
9. Remove the supernatant, resuspend with 1 mL of binding buffer.

# X-ray photoelectron spectroscopy (XPS) Analysis

# Equipment

1. Diamond glass cutter
2. 250 mL beaker
3. Ultrasonic cleaner
4. Oven
5. Vacuum dryer
6. High resolution X-ray Photoelectron Spectrometer (ULVAC-PHI, PHI Quantera Ⅱ)

# Consumables

1. Silicon wafer
2. Dish detergent
3. Ethanol
4. Acetone
5. Petri dish

# Protocol

**Sample Preparation**

1. Cut the silicon wafer to approximately 0.8 cm\*0.8 cm square with a diamond glass cutter.
2. Put the silicon wafer in a clean beaker. Wash the silicon wafer with diluted dish detergent
in an ultrasonic cleaner for 12 minutes.
3. Transfer the silicon wafer to another clean beaker. Wash the silicon wafer with ddH2O in an
ultrasonic cleaner for 12 minutes. Repeat this step three times.
4. Transfer the silicon wafer to another clean beaker. Wash the silicon wafer with ethanol in an
ultrasonic cleaner for 12 minutes.
5. Transfer the silicon wafer to another clean beaker. Wash the silicon wafer with acetone in an
ultrasonic cleaner for 12 minutes.
6. Transfer the silicon wafer to another clean beaker. Wash the silicon wafer with ddH2O in an
ultrasonic cleaner for 12 minutes.
7. Transfer it to a clean Petri dish. Dry it in an oven.
8. Drop 30 µL of nanoparticles sample on a silicon wafer.
9. Put it in a vacuum dryer for 1-2 hours, confirm the sample is completely dry.
10. Drop 30 µL of nanoparticles sample on a silicon wafer.
11. Put it in a vacuum dryer for 1-2 hours, confirm the sample is completely dry.

**Analysis**

1. Using High resolution X-ray Photoelectron Spectrometer (ULVAC-PHI, PHI Quantera Ⅱ) to analyze
the elemental composition and distribution in the sample.

# RCA

# Using ProtoScript® II Reverse Transcriptase

# Equipment

1. Thermocycler

# Consumables

1. ssRNA ladder (500-9000 bp) (New England Biolabs)
2. Primer 1 (50 μM)
3. Primer 2 (50 μM) **\*Note: We adopted two primers which differ in sequence and thus the binding
site on the
circRNA, with the attempt to increase the efficiency of RCA.**
4. dNTP (10 μM) (Thermo Fisher Scientific)
5. 5X ProtoScript II Reaction Buffer (New England Biolabs)
6. ProtoScript® II Reverse Transcriptase (200 U/µL) (New England Biolabs)
7. 0.1 M DTT (Thermo Fisher Scientific)
8. RNase Inhibitor (40 U/mL) (New England Biolabs)
9. Sample 1 (circRNA) (~50 ng)
10. Sample 2 (IVT product, linear RNA) (~50 ng)
11. Nuclease-free water (Protech Technology)

# Protocol

1. Add the following components. (We employed products generated from circularization and IVT to
compare the differences between RCA products of circular and linear RNAs, by observing gel
electrophoresis result.)

|  |  |  |
| --- | --- | --- |
| Reagents | Amount | Amount |
| Sample | CircRNA (variable) | Linear RNA (variable) |
| Primer 1 | 2 μL | 2 μL |
| Primer 2 | 2 μL | 2 μL |
| dNTP | 1 μL | 1 μL |
| Nuclease-free water | to 10 μL | to 10 μL |
| Total | 10 μL | 10 μL |

2. Denature sample RNA & primers for 5 minutes at 65 °C. Spin briefly and put promptly on ice.
3. Add the following components to the denatured reaction.

|  |  |
| --- | --- |
| Reagents | Amount |
| 5X ProtoScript 2 RT Reaction Buffer | 4 μL |
| 0.1 M DTT | 2 μL |
| ProtoScript II RT | 1 μL |
| RNase Inhibitor | 0.2 μL |
| Nuclease-free water | to 10 μL |
| Total | 10 μL |

4. Incubate the 20 μL RCA reaction in a thermocycler at the following settings.

|  |  |
| --- | --- |
| Temperature | Time |
| 42 °C | 60 min |
| 65 °C | 20 min |
| 4 °C | ∞ |

\*\*Note: React at 42 °C for one hour, then inactivate the enzyme at 65 °C for 20
minutes.\*\*

5. Pipet 1 μL ssRNA ladder and 9 μL RNA loading dye in a PCR tube, incubate at 90 °C for 2
minutes, then put it on ice for 1~2 mins.
6. Mix 15 μL RCA product with 3 μL dye.
7. Run gel electrophoresis to confirm the size.
8. The remaining cDNA product should be stored at -20 °C.

# Using Induro® Reverse Transcriptase

# Equipment

1. Thermocycler

# Consumables

1. Primer 1 (50 μM)
2. Primer 2 (50 μM) **\*Note: We adopted two primers which differ in sequence and thus the binding
site on the
circRNA, with the attempt to increase the efficiency of RCA.**
3. dNTP (10 mM) (Thermo Fisher Scientific)
4. Induro® RT Reaction Buffer (New England Biolabs)
5. Induro® Reverse Transcriptase (200,000 U/mL) (New England Biolabs)
6. RNase inhibitor (New England Biolabs)
7. ssRNA ladder (New England Biolabs)
8. RNA loading dye (New England Biolabs)
9. Nuclease-free water (Protech Technology)
10. Sample (circRNA) (~50 ng)
11. Sample (IVT product, linear RNA) (~50 ng)

# Protocol

1. Add the following components. (We employ products generated from circularization and IVT to
compare the differences between RCA products of circular and linear RNAs, by observing gel
electrophoresis result.)

|  |  |  |
| --- | --- | --- |
| Reagents | Amount | Amount |
| Sample | CircRNA (variable) | Linear RNA (variable) |
| Primer 1 | 2 μL | 2 μL |
| Primer 2 | 2 μL | 2 μL |
| dNTP | 1 μL | 1 μL |
| Nuclease-free water | to 10 μL | to 10 μL |
| Total | 10 μL | 10 μL |

2. Denature at 65 °C, 5 min and keep on ice for 1~2 mins.
3. Add the following components to the denatured reaction.

|  |  |
| --- | --- |
| Reagents | Amount |
| 5X ProtoScript 2 RT Reaction Buffer | 4 μL |
| RNase inhibitor | 0.2 μL |
| Induro RT | 1 μL |
| Nuclease-free water | 4.8 μL |
| Total | 10 μL |

4. Incubate at 60 °C, 15 mins, then cool down at 4 °C.
5. Pipet 1 μL ssRNA ladder and 9 μL RNA loading dye into a PCR tube, incubate at 90 °C for 2
minutes, then put it on ice for 1~2 mins.
6. Mix 15 μL RCA product with 3 μL dye.
7. Run agarose gel electrophoresis to check the size of the RCA product.
8. The cDNA product should be stored at -20 °C.

# Probe conjugated-AuNP detection

# Equipment

1. SPECTROstar Nano (UV/Vis spectrometer)
2. Camera (smartphone)
3. Cuvettes

# Consumables

1. 0.21 M MgCl2
2. RCA solution with no target
3. RCA solution with target (circRNA)

# Protocol

**RCA solution with no target**

1. Dilute probe conjugated-AuNP solution O.D. to 2.56. Conduct UV-Vis spectroscopy by SPECTROstar
Nano.
2. Pipet 180 µL of probe conjugated-AuNP solution into a cuvette. Add 20 µL of RCA with NTC (no
template control) solution. Use SPECTROstar Nano to conduct UV-Vis spectroscopy to compare the
probe-conjugated AuNP solution with the one without the addition of RCA solution (with no
target). Confirm that there was no redshift in the spectrum or aggregation of the gold
nanoparticles.
3. Add 1 µL 0.21 M MgCl2, react for 2 minutes during each interval. Take a photo and
conduct
UV-Vis spectroscopy by SPECTROstar Nano.
4. Repeat step 3 for multiple times until the solution undergoes a noticeable color change
and a
redshift in the spectrum. Record the volume of MgCl2 added.

**RCA solution with target**

1. Dilute probe conjugated-AuNP solution O.D. to 2.56. Conduct UV-Vis spectroscopy by SPECTROstar
Nano.
2. Pipet 180 µL probe conjugated-AuNP solution to a cuvette. Add 20 µL RCA solution containing
RCA amplicons. Use SPECTROstar Nano to conduct UV-Vis spectroscopy to compare the probe
conjugated-AuNP solution with the one without addition of RCA solution. Confirm that there was
no redshift in the spectrum or aggregation of the gold nanoparticles.
3. Add the same volume of 0.21 M MgCl2 in the "RCA solution with no target" experiment.
Let it
react for 2 minutes. Take a photo and conduct UV-Vis spectroscopy by SPECTROstar Nano.
4. Confirm the target exists or not by observing the color change after adding nanoparticles
and
MgCl2 to the RCA solution.

# Repeated insert sequence synthesis

# Insert digestion

# Equipment

1. Dry bath

# Consumables

1. 10X tangle buffer (Thermo Fisher Scientific)
2. Restriction enzyme: BamH1 (10 U/µL) (Thermo Fisher Scientific)
3. Restriction enzyme: HindIII (10 U/µL) (Thermo Fisher Scientific)
4. Nuclease free water (Protech Technology)
5. Sample (insert)
6. Gel/PCR DNA Isolation System (VIOGENE)

# Protocol

1. Preheat dry bath to 37 °C
2. Set up the digestion reaction on ice. Vortex briefly and spin down for 3-5 s.

|  |  |
| --- | --- |
| Reagents | Amount |
| 10X tangle Buffer | 10 μL (2X ends) |
| Sample | ~1 μg |
| BamH1 | 1 μL |
| HindIII | 2 μL |
| Nuclease-free water | to 50 μL |
| Total | 50 μL |

3. Incubate the digestion mixture at 37 °C for 1 hr.
4. Run the digested DNA on a 1.5 % agarose gel.
5. Conduct gel purification using Gel/PCR DNA Isolation System followed the provided
protocol.
6. Measure a DNA concentration of the purified product by NanoDrop. (About 50 ng is needed for
the following procedure)
7. Use the purified product directly for ligation.

# Insert ligation

# Equipment

NA

# Consumables

1. Digestion product
2. T4 DNA ligase buffer (10X) (New England Biolabs)
3. T4 DNA ligase (New England Biolabs)
4. Sample (Insert)
5. Gel/PCR DNA Isolation System (VIOGENE)

# Protocol

1. Set up the ligation reaction on ice. Vortex briefly and spin down for 3-5 s.

|  |  |
| --- | --- |
| Reagents | Amount |
| T4 DNA ligase buffer (10X) | 2 μL (1X ends) |
| Sample | ~50 ng |
| T4 DNA ligase | 1 μL |
| Nuclease-free water | to 20 μL |
| Total | 20 μL |

2.Incubate at room temperature (25 °C) overnight.
Notice: Since the desired product is about 3000 bp, the incubation time needs to be prolonged
(overnight) for better yields.
3. Run the ligation product on a 1.2 % agarose gel.
4. Conduct gel purification using Gel/PCR DNA Isolation System followed the provided
protocol.
5. Measure a DNA concentration of the purified product by NanoDrop spectrophotometer.
6. Store the insert ligation product at 4 °C.

# Preparation of template for AELA-PCR

# Ligation with vector

# Vector Digestion

# Equipment

1. Dry bath

# Consumables

1. 10X tangle buffer (Thermo Fisher Scientific)
2. Restriction enzyme: BamH1 (10 U/µL) (Thermo Fisher Scientific)
3. Restriction enzyme: HindIII (10 U/µL) (Thermo Fisher Scientific)
4. Nuclease-free water (Protech Technology)
5. pUC19 vector (New England Biolabs)

# Protocol

1. Preheat dry bath to 37 °C
2. Set up the digestion reaction on ice. Vortex briefly and spin down for 3-5 s.

|  |  |
| --- | --- |
| Reagents | Amount |
| 10X tangle buffer | 10 μL (ends in 2X) |
| pUC19 vector | 0.04 pmol |
| BamH1 | 1 μL |
| HindIII | 2 μL |
| Nuclease-free water | to 50 μL |
| Total | 50 μL |

3. Incubate the digestion mixture at 37 °C for 1 hr.
4. Use the digest vector directly for ligation.

# Vector ligation

# Equipment

NA

# Consumables

1. Vector digestion product
2. Sample (insert ligation product)
3. T4 DNA ligase buffer (10X) (New England Biolabs)
4. T4 DNA ligase (New England Biolabs)
5. Gel/PCR DNA Isolation System (VIOGENE)
6. Nuclease-free water (Protech Technology)

# Protocol

1. Set up the ligation reaction on ice. Vortex briefly and spin down for 3-5 s.

|  |  |
| --- | --- |
| Reagents | Amount |
| 10X T4 DNA ligase buffer | 2 μL (2X ends) |
| Vector digestion product | 0.04 pmol ends |
| Insert ligation product | 0.04 pmol ends |
| T4 DNA ligase | 1 μL |
| Nuclease-free water | to 20 μL |
| Total | 20 μL |

2. Incubate at room temperature (25 °C) overnight.
3. Store the insert ligation product at 4 °C or directly use for transformation.

# Transformation

# Equipment

1. Dry bath
2. Petri dish
3. Disposable cell spreaders
4. Incubator

# Consumables

1. DH5-alpha E. coli
2. Sample (ligation with vector product)
3. LB Medium plate with ampicillin (0.1 mg ampicillin / 1 mL LB)

# Protocol

1. Preheat the dry bath to 42 °C.
2. Add 5 μL ligation product into 50 μL DH5-alpha E.coli stock. Do not pipet or vortex, flick
the tube slightly about 5 times.
3. Put the reaction on ice for 2 min.
4. Incubate at 42 °C for precisely 30 sec.
5. Put on ice for 2 min.
6. Add 950 μL cell culture medium.
7. Take 100 μL of the mixture and spread it evenly on a plate using a Disposable cell
spreaders.
8. Incubate the plate in the incubator at 30 °C for 24 hr.

# Colony PCR

# Equipment

1. Culture tube
2. PCR tube
3. Thermocycler

# Consumables

1. LB medium with ampicillin (0.1 mg ampicillin / 1 mL LB)
2. 2X Master Mix (Protech Technology)
3. PCRforAELA\_fw primer, 10 μM
4. PCRforAELA\_rv primer, 10 μM
5. Sample (transform product)
6. Gel extraction system

# Protocol

1. Use pipette tips to pick colony, each Petri dish choose 2 colonies
2. Put the colony in 200 μL of LB with ampicillin (1 μL ampicillin/1mL LB)
3. Shake in a 37°C incubator for 2 hours in preparation for colony PCR.
4. Add the following reagent to PCR tube

|  |  |
| --- | --- |
| Reagents | Amount |
| 2X Master Mix | 10 μL (1X ends) |
| Free water | 7 μL |
| Foward primer | 0.5 μL |
| Reverse primer | 0.5 μL |
| Sample | 2 μL |
| Total | 20 μL |

5. Conduct PCR at following thermal cycle.

|  |  |  |  |
| --- | --- | --- | --- |
| Stage | Temperature | Time |  |
| Denaturation | 94 °C | 1 min |  |
| Denaturation | 94 °C | 30 sec | 35 cycles |
| Annealing | 61 °C | 30 sec |
| Extension | 72 °C | 1 min 5 sec |
| Extension | 72 °C | 7 min |  |
| Cooling | 4 °C | ∞ |  |

6. Run the PCR product on a 1.2 % Agarose Gel.
7. We found out that there was no band presented on the agarose gel, we want to purify the
plasmid to run gel electrophoresis instead. To extract plasmid, transfer 10 μL bacteria liquid
to 5 mL LB with ampicillin in a culture tube first.
8. Shake in a 37 °C incubator for 16 hours.

# Plasmid extraction

# Equipment

1. Centrifuge tube

# Consumables

1. Mini Plus Plasmid DNA Extraction System (Viogene)

# Protocol

1. Perform plasmid extraction follow the protocol provided by the manufacturer.
2. Run the extracted plasmid on a 1.2 % agarose gel to identify whether the ligation process was
successful.

# AELA-PCR

# Equipment

1. PCR tubes
2. Thermocycler

# Consumables

1. i-Taq 2X PCR master mix kit (Intron Biotechnologies)
(the kit contains 2.5 U/μL of DNA polymerase, 2.5 mM each of dNTPs, and 1X reaction buffer with
3 mM MgCl2)
2. Forward primer (0.2 μM)
3. Reverse primer (10 μM) **\*Note: The concentration ratio of the extended (rv) primer to
un-extended (fw) primer should be 20:1. ([See Parts\_AELA\_PCR](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27parts%27%29%20%7D%7D))**
4. Nuclease-free water (Protech Technology)
5. Sample (long-repeated dsDNA)

# Protocol

1. Add the following reagents to PCR tube.

|  |  |
| --- | --- |
| Reagents | Amount |
| i-Taq mix | 10 μL |
| Primer mix (extended primer:un-extended primer = 20:1) | 1 μL |
| Long-repeated dsDNA sample | 1 μL |
| Nuclease-free water | 8 μL |
| Total | 20 μL |

2. Conduct PCR at following thermal cycle.
3. React in a thermocycler at following setting.

|  |  |  |  |
| --- | --- | --- | --- |
| Stage | Temperature | Time |  |
| Denaturation of dsDNA | 95 °C | 2 min |  |
| Exponential phase | 95 °C | 20 sec | 35 cycles |
| 58 °C | 15 sec |
| 72 °C | 40 sec |
| Linear phase | 95 °C | 20 sec | 25 cycles |
| 72 °C | 50 sec |
| Forming ssDNA amplicons | 72 °C | 2 min |  |

4. Conduct agarose gel electrophoresis to confirm our PCR products.

# RPA

# Equipment

1. 8 Low Profile PCR tube
2. Thermocycler
3. Mini centrifuge

# Consumables

1. Rehydration buffer (TwistDx)
2. Forward primer (10 µM)
3. Reverse primer (10 µM)
4. MgOAc (280 mM) (TwistDx)
5. RNAse-free water (Protech Technology)
6. Sample (insert)
7. TwistAmp® Basic reaction pellets tubes (TwistDx)

# Protocol

1. Add the following reagents to TwistAmp® Basic reaction pellets tubes.

|  |  |
| --- | --- |
| Reagents | Amount |
| Primer free rehydration buffer | 29.5 μL |
| Foward primer | 2.4 μL |
| Reverse primer | 2.4 μL |
| Sample | ~100 ng |
| RNase-free water | to 47.5 μL |
| Total | 47.5 μL |

\*\*Note: Forward primer and reverse primer are designed by ourselves. For more
details, please view [Parts\_RPA primer.\*\*](#{{ url_for('pages', page='parts') }}#36019)

2. Add 2.5 µL of 280 mM MgOAc to the lid of TwistAmp® Basic reaction pellets tubes.
3. Spin down with a mini centrifuge to introduce MgOAc into the rehydrated material and initiate
the reactions.
4. Vortex briefly and spin down once again.
5. Incubate the tubes in thermocycler under 37˚C for 20 minutes.
6. After 20 minutes, run gel electrophoresis to confirm if the amplification is successful. For
the lateral flow test, follow the prescribed instructions to appropriately dilute the reaction
products.

# RT-RPA

# Equipment

1. Thermocycler

# Consumables

1. Primer free rehydration buffer (TwistDx)
2. Forward primer (10 µM)
3. Reverse primer (10 µM)
4. ProtoScript® II Reverse Transcriptase (200 U/µL) (New England Biolabs)
5. RNase inhibitor (New England Biolabs)
6. dNTP (10 mM) (Thermo Fischer Scientific)
7. RNase free water (Protech Technology)
8. Sample (IVT product, linear RNA)

# Protocol

1. Prepare reaction mix in 1.5 mL tube

|  |  |
| --- | --- |
| Reagents | Amount |
| Primer free rehydration buffer | 29.5 μL |
| Foward primer | 2.4 μL |
| Reverse primer | 2.4 μL |
| Protoscript RT | 0.5 μL |
| RNase inhibitor | 0.5 μL |
| dNTP | 1 μL |
| Sample | 2 μL |
| RNase-free water | to 50 μL |
| Total | 50 μL |

2. Add reaction mix to a TwistAmp® Basic reaction. Pipette to mix.
3. Add 2.5 μL of 280 mM Magnesium Acetate (MgOAc) and mix well to start the reaction. **\*Note: RPA
reactions start as soon as MgOAc is added.**
4. Incubate at 39 ˚C for 20 minutes.
5. After 20 minutes, run gel electrophoresis to confirm if the amplification is successful. For
the lateral flow test, follow the prescribed instructions to appropriately dilute the reaction
products.

# PCRD

# Equipment

NA

# Consumables

1. PCRD (lateral flow strip cassette) (Abingdon Health)
2. PCRD Extraction Buffer (Abingdon Health)
3. Sample (RT-RPA product)

# Protocol

1. Pipette 6 µL of the amplification product into a 0.5 mL tube.
2. Transfer 84 µL of the PCRD Extraction Buffer supplied into the tube and mix thoroughly.
3. Open the foiled PCRD, add 75 µL of the diluted reaction mixture to the sample well of a PCRD
test cassette.
4. Leave the cassette in a horizontal position for 10 mins while the result develops. Read the
result visually at 10 mins. Ignore any changes which occur after 10 mins.

# Reference

[1]
<https://www.thermofisher.com/order/catalog/product/EF022SKAFB001?ef_id=Cj0KCQjwnMWkBhDLARIsAHBOftoRPvAmH5H5ades6_zg-NfxauPrhK4FU7fLkJR8HwkrpuzhgW_EWcEaApmfEALw_wcB:G:s&s_kwcid=AL!3652!3!635884956165!p!!g!!pyrophosphatase!17547406734!147243632801&cid=bid_nat_mrn_r01_co_cp0000_pjt0000_bid00000_0se_gaw_nt_awa_awa&gclid=Cj0KCQjwnMWkBhDLARIsAHBOftoRPvAmH5H5ades6_zg-NfxauPrhK4FU7fLkJR8HwkrpuzhgW_EWcEaApmfEALw_wcB>

[2]
<https://www.thermofisher.com/order/catalog/product/EN0521>

[3]
<https://www.abcam.com/products/sample-preparation-kits/rnase-r-ab286929.html>

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block page\_content %}

Results

* [Path A](#Path A)

+ [Cloning](#Cloning)
+ [PCR](#PCR)
+ [In vitro transcription (IVT)](#In vitro transcription (IVT))

* [Path B](#Path B)

+ [Gold
  nanoparticle (AuNPs) synthesis](#Gold nanoparticle (AuNPs) synthesis)
+ [Probe
  conjugated with
  gold nanoparticles (AuNPs)](#Probe conjugated with gold nanoparticles (AuNPs))
+ [Probe
  conjugated with Gold nanoparticle (AuNPs) detection](#Probe conjugated with Gold nanoparticle (AuNPs) detection)

* [Path C](#Path C)

+ [Repeated insert sequence
  synthesis](#Repeated insert sequence synthesis)
+ [Preparation of template
  for
  AELA-PCR](#Preparation of template for AELA-PCR)
+ [AELA PCR](#AELA PCR)

* [Path D](#Path D)

+ [Rolling
  Circle Amplification (RCA)](#Rolling Circle Amplification (RCA))
+ [Probe
  conjugated with gold nanoparticles (AuNPs) detection](#Probe conjugated with gold nanoparticles (AuNPs) detection)

* [Path E](#Path E)

+ [Recombinase
  Polymerase
  Amplification (RPA)](#Recombinase Polymerase Amplification (RPA))
+ [PCRD](#PCRD)

* [Path F](#Path F)

+ [RT-RPA](#RT-RPA)
+ [PCRD](#PCRD2)

* [Path G](#Path G)
* [Reference](#Reference)

# Path A: CircRNA Synthesis

**Sequence design → Cloning → PCR → In vitro transcription (IVT) → Circularization → CircRNA
enrichment**

# Cloning

# Description

In this part, we ligase our insert with pJET1.2/blunt
Cloning Vector and then transform it in
DH5-alpha E. coli in order to produce abundant amounts of our target.

# Data

![](https://static.igem.wiki/teams/4636/wiki/results-circrna-synthesis/figure-a1.jpg)

Figure A1. Gel electrophoresis results of the colony PCR. lane 1: Bio-100 Mass DNA Ladder, lane
2: circ\_0004771 colony 1, lane 3: circ\_0004771 colony 2, lane 4: circ\_0004771 colony 3, lane 5:
circ\_0004771 colony 4, lane 6: x, lane 7: x, lane 8: circ\_0101802 colony 1, lane 9: circ\_0101802
colony 2, lane 10: circ\_0101802 colony 3, lane 11: circ\_0101802 colony 4

Figure A1 shows the gel electrophoresis results of the
colony PCR of the transformation product.
We can find out that lane 4, 5, 9, and 10 have no unwanted bands, so we culture them and extract
their plasmid for sequencing.

![](https://static.igem.wiki/teams/4636/wiki/results-circrna-synthesis/figure-a2.png)

Figure A2. Sequence alignment of circ\_0004771 colony 3 PCR product with circ\_0004771 insert.

![](https://static.igem.wiki/teams/4636/wiki/results-circrna-synthesis/figure-a3.png)

Figure A3. Sequence alignment of circ\_0004771 colony 4 PCR product with circ\_0004771 insert.

![](https://static.igem.wiki/teams/4636/wiki/results-circrna-synthesis/figure-a4.png)

Figure A4. Sequence alignment of circ\_0101802 colony 3 PCR product with circ\_0101802 insert.

![](https://static.igem.wiki/teams/4636/wiki/results-circrna-synthesis/figure-a5.png)

Figure A5. Sequence alignment of circ\_0101802 colony 4 PCR product with circ\_0101802 insert.

The sequencing results show that our product sequence is
99% similar to our insert, which indicates the success of the colony and it can be used for
further experiments.

# Achievement

Successfully ligase insert the vector and transform it into
DH5-alpha E. coli.

# PCR

# A. Insert PCR

# Description

Insert PCR is the method we use for
the rapid and large-scale production of Insert\_0101802 and Insert\_0004771. The resulting
products can be used for circularization and experiment validation in other sections.

# Data

![](https://static.igem.wiki/teams/4636/wiki/results-circrna-synthesis/figure-a6.png)

Figure A6. PCR result of Insert\_0004771. Lane 1 : 100bps ladder, Lane 2 : Insert\_0004771

![](https://static.igem.wiki/teams/4636/wiki/results-circrna-synthesis/figure-a7.jpg)

Figure A7. PCR result of Insert\_0101802.Lane 1 : 50bps ladder, Lane 2,3 : Insert\_0101802

# Analysis

As shown in the Figure A6 and
Figure A7, bright clear bands in the correct position can be seen, which indicates the
successful amplifaction of Insert\_0101802 and Insert\_0004771 by Insert PCR.

# B. Plasmid PCR

# Description

Plasmid is produced in large
quantities by DH5-alpha E. coli after cloning. In this part, plasmid is extract from the
bacteria (for detailed please visit experiment-Plasmid extraction) and futher conduct plasmid
PCR in order to rapidly produce large-scale production of Insert\_0101802 and Insert\_0004771. The
resulting products can be used for circularization and experiment validation in other sections.

# Data

![](https://static.igem.wiki/teams/4636/wiki/results-circrna-synthesis/figure-a8.jpg)

Figure A8. Plasmid PCR result. Lane 1 : 100bps ladder, Lane 2, 4 : pjet1.2\_0004771 , Lane 3, 5 :
PCR of pjet1.2\_0004771, Lane 6, 8 : pjet1.2\_0101802 , Lane 7, 9 : PCR of pjet1.2\_0101802

# Analysis

As shown in the figure, we have
successfully amplified pjet1.2\_0101802 and pjet1.2\_0004771 using Plasmid PCR. We will further
purify by gel slices and us for further experiment.

# CircRNA synthesis (IVT & circularization & CircRNA enrichment)

In this process, we utilize
the Insert 0101802 (Part) to undergo in vitro transcription (IVT), circularization, and circRNA
enrichment. Due to time and resource constraints, circularizing two samples at once is not very
efficient. Therefore, we use Insert 0101802 to initially validate the entire circRNA synthesis
process's feasibility. Once we confirm the viability of the entire process, we can apply it to
all samples to achieve our goal.

A. Verification of
monophosphorylation and circularization product

# Description

# **XRN-1 - verification of monophosphorylation**

In order to verify the success of monophosphorylation, we
utilize an enzyme, XRN-1. XRN-1 is an
exoribonuclease enzyme that could degrade RNA sequences with a 5' monophosphate group.
Therefore, RNA that is successfully monophosphorylated could be disintegrated by XRN-1. We run
E-gel to compare monophosphorylation product with monophosphorylation product + XRN-1. If the
monophosphorylation product + XRN-1 has a band as clear as the monophosphorylation product, then
monophosphorylation is failed. Conversely, if the band is less distinct than that of the
monophosphorylation product or no band is observed at all, it signifies successful
monophosphorylation

# **RNase R - Eliminate linear form RNA**

After circularization process, we are supposed to transform
all the linear form RNA into circRNA. However, research related to circularization has not yet
achieved 100% circularization rate. Therefore, in order to obtain pure circRNA, we use RNase R
to eliminate failed circularization products, which are linear RNAs. After RNase R degrades the
linear RNA, circRNA remains.

![](https://static.igem.wiki/teams/4636/wiki/results-circrna-synthesis/figure-a9.jpg)

Figure A9. Verification of monophosphorylation and circularization product. Lane M: low range
RNA ladder, Lane 1: Linear form RNA, Lane 2: Monophosphorylation product, Lane 3:
Monophosphorylation product + XRN-1, Lane 4: Circularization product, Lane 5: Circularization
product + RNase R, Lane 6: Circularization product + XRN-1, Lane7-10: Negative Control

# Analysis

1. Lane1: Lane 1 is the sample of IVT product. After IVT, we obtained linear form RNA, as the
above figure shows.

2. Lane 2 and Lane 3: Comparing lane 2 and lane 3, lane 2 obtained a brighter band than lane
3.
After adding XRN-1, the monophosphorylated product has been degraded, leaving failed
monophosphorylated samples. Therefore, the remaining band of lane 3 indicates the RNAs that have
failed to be monophosphorylated. In summary, by contrast of these two lanes, we know that the
efficiency of monophophorylation isn't 100%; there's still room for improvement.

3. Lane 4 and Lane 5: Comparing lane 4 with lane 5, lane 4 obtained a brighter band than
lane 5.
After adding RNase R to the circularized product, linear-form RNA could be degraded, leaving RNA
with a circular structure. Therefore, lane 5 represents lane 4 sample eliminating linear form
RNA, which means lane 5 are samples successfully circularized.

4. Lane 6: In order to reconfirm the success of circularization, we add XRN-1 to
Circularization
product. In circRNA, there's no monophosphorylated end to be degraded by XRN-1. Therefore,
successfully circularized product should remain band after adding XRN-1.

# Improvement

Sample bands in
this
E-gel experiment are slightly smear. We suspected that this smear phenomenon is caused by
incomplete denature of samples. As a result, we revise denature condition from 65°C, 5 mins to
65°C, 15 mins.

# Achievement

After conducting
this
experiment, we verify success of monophosphorylation and circularization, which proof that we're
capable of synthesizing circRNA.

B. Comparing circularization efficiency of splint with different length

# Description

In order to
compare
circularization efficiency of splint with different length, we carry out circRNA synthesis
process using 14-mer splint, 16-mer splint, 18-mer splint, respectively. After running
E-gel,
splint with highest circularization efficiency obtained strongest band. Through this
verification, we could utilize the splint with highest circularization efficiency in the
following process.

![](https://static.igem.wiki/teams/4636/wiki/results-circrna-synthesis/figure-a10-png.jpg)

Figure A10. Comparing circularization efficiency of splint with different length. Lane 1: low
range RNA ladder, Lane 2: Control, Lane 3: Circularization product using 14-mer splint, Lane 4:
Circularization product using 16-mer splint, Lane 5: Circularization product using 18-mer splint

# Analysis

Lane 3, Lane 4, and Lane 5: Comparing lane 3,
lane 4, and lane 5, we discovered
that lane 4 has the strongest band. It indicates that using a 16-mer splint could maximize
circularization yield.

# Achievement

After conducting this experiment, we confirm
utilizing 16-mer splint could
maximize circularization yield.

C. Distinguish circRNA from linear form RNA

# Description

Previous studies have mentioned that in gel electrophoresis, circRNA has
higher running speed comparing to linear form RNA. This conclusion made use curious that whether
our circularization product and linear form RNA could distinguish by their band position. Also,
since we could merely distinguish band of ladder in E-gel results, we tried agarose gel instead.
By utilizing agarose gel, we could clearly compare our sample bands to ladder band, for
confirming size of samples.

![](https://static.igem.wiki/teams/4636/wiki/results-circrna-synthesis/figure-a11-png.jpg)

Figure A11. Distinguish circRNA from linear form RNA. Lane 1: low range RNA ladder, Lane 2:
Linear form RNA, Lane 3: Circularization product, Lane 4: Circularization product + RNase R,
Lane 5: Circularization product + RNase R

# Analysis

1. Lane 2 ~ Lane 5: According to the
low-range RNA ladder, the size of the
samples could be confirmed. IVT product, circularization product, and circularization product +
RNase R are all around 300 bp, which conforms to our assumption.

2. Lane 2, Lane 4, Lane 5: Comparing lane 2 and lane 4, 5, we discovered that lane 2 band is
higher than lane 4, 5. Previous research have mentioned that circular form RNA runs faster than
linear form RNA. Since they obtained the same base pair, we suspect that it's the structure that
affects their running speed. As a result, we conclude that Lane 4, Lane 5 is certainly circular
RNA.

# Achievement

After conducting this experiment, we confirm
that all our samples obtained
correct base pair as we expected. Furthermore, by comparing position of linear form RNA and
circularization product, we verify the success of circularization.

D. Examine RNase R degradation ability

# Description

In order to proof the degradation ability of
RNase R is powerful enough to
eliminate all the failed circularization product. We conduct following experiment, adding RNase
R into linear form RNA product from in vitro transcription (IVT) process. We expected that RNase
R could effectively degrade entire linear form RNA, which confirmed that it could also degrade
entire failed circularization product after circRNA synthesis process.

![](https://static.igem.wiki/teams/4636/wiki/results-circrna-synthesis/figure-a12.jpg)

Figure A12. Examine RNase R degradation ability. Lane M: low range RNA ladder, Lane 1: Linear
form RNA, Lane 2: Linear form RNA + RNase R, Lane 3: Circularization product + RNase R, Lane
4-10: Negative Control

# Analysis

1. Lane 1 and Lane 2: In order to make sure the degradation ability of RNase R is powerful
enough to eliminate entire linear RNA in a circularized product, we add RNase R to IVT samples.
After adding RNase R, we discovered that there's no band in lane 2, which indicates that RNase R
has degraded the entire linear form of RNA. This result confirmed that RNase R could effectively
eliminate redundant linear-form RNA in circularization samples, leaving pure circRNA.

2. Lane 3: After adding RNase R to the circularization product, there's a remaining band.
This
band is a sample that has been successfully circularized.

# Achievement

After conducting this experiment, we confirm degradation ability of RNase R is powerful enough
to eliminate all the failed circularization product

# Path B: Probe-Conjugated Gold Nanoparticle Synthesis

**Sequence design → Gold nanoparticle (AuNPs) synthesis → Probe conjugated with Gold
nanoparticle (AuNPs) → Probe conjugated with gold nanoparticle (AuNPs) detection**

# Gold nanoparticle (AuNPs) synthesis

# Description

To utilize a colorimetric assay for
detecting colorectal cancer, our initial step involves synthesizing gold nanoparticles (AuNPs)
of approximately 13 nm in diameter. Once we have successfully synthesized it, we can then
proceed to conjugating probes onto these synthesized gold nanoparticles.

# Data

![](https://static.igem.wiki/teams/4636/wiki/results-colorimetric-assay/figure-b1.jpg)

Figure B1. Synthesis of gold nanoparticles in a two-neck flask

![](https://static.igem.wiki/teams/4636/wiki/results-colorimetric-assay/figure-b2.jpg)

Figure B2. The spectrum of the gold nanoparticles exhibits a peak that is close to 520nm.

![](https://static.igem.wiki/teams/4636/wiki/results-colorimetric-assay/figure-b3.jpg)

Figure B3. Examine gold nanoparticles under a scanning electron microscope (SEM) using the
following conditions: voltage: 1500 kV, magnification: 230,000 folds.

# Analysis

In previous studies, we discovered that 13 nm gold
nanoparticles typically exhibit a peak at 520
nm in the spectrum[[1]](#[1]). To validate whether our self-synthesized gold nanoparticles are around 13
nm, we conducted a spectrum measurement. The results, shown in Figure B2, indicated a peak that
closely aligns with 520 nm, providing strong evidence of successful synthesis. Additionally, we
utilized a Scanning Electron Microscope (SEM) to examine gold nanoparticles. The results, shown
in Figure B3, directly revealed that the gold nanoparticles have an approximate diameter of 13
nm, confirming that we have successfully synthesized gold nanoparticles of the desired size.

# Achievement

1. Successfully synthesized 13 nm gold nanoparticles.

# Probe conjugated with gold nanoparticles (AuNPs)

# A. Nanodrop Spectrum Analysis

# Description

In this section, the probe will be conjugated with gold
nanoparticles through an S-Au bond. Our probe is designed to detect the target cDNA generated by
RCA. For more detailed design information, please refer to the Parts page.

# Data

![](https://static.igem.wiki/teams/4636/wiki/results-colorimetric-assay/figure-b4.png)

Figure B4 . A redshift shown in the spectrum of gold nanoparticles after conjugated with probes.

# Analysis

Figure B4 revealed a redshift occurring in the spectrum of
gold nanoparticles after conjugated with probes. According to previous studies [[2]](#[2]), the redshift
in the spectrum and a decrease in the optical density (O.D.) value are indicative of the
successful conjugation of AuNPs with the probe.

# Achievement

Successfully attached DNA probes to the surface of gold
nanoparticles.

# B. X-ray photoelectron spectroscopy (XPS) Analysis

# Description

In addition to verifying the successful attachment of the
probe to gold nanoparticles, we also employed X-ray Photoelectron Spectroscopy (XPS) to examine
the presence of DNA probes on the surface of the gold nanoparticles. This involved examining
elements associated with DNA probe and gold nanoparticles, such as Au, C, and N, and comparing
the differences before and after probe modification on gold nanoparticles.

![](https://static.igem.wiki/teams/4636/wiki/results-colorimetric-assay/figure-b5.jpg)

Figure B5. XPS results of gold nanoparticles and probe-conjugated gold nanoparticles.

# Achievement

Successfully attached DNA probes to the surface of gold
nanoparticles.

# Probe conjugated with Gold nanoparticle (AuNPs) detection

# Description

Probe conjugated with gold
nanoparticles will be used to conduct further detection of long reapeat cDNA sequence. For more
detailed, please view Path C and Path D.

# Path C: Assess the Functionality of Colorimetric Assay with AELA-PCR

**Sequence design → Cloning → PCR → Repeated insert sequence synthesis → Preparation of
template for AELA-PCR → AELA-PCR → Probe conjugated AuNPs detection**

# Repeated insert sequence synthesis

Description: We have added two restriction enzymes, BamH1 and HindIII, to cleave the PCR
products. The resulting sticky ends at the 3' and 5' end of the inserts were then joined
together using T4 DNA ligase, creating long-repeated insert sequences.

# A. Insert digestion

# Data

![](https://static.igem.wiki/teams/4636/wiki/results-colorimetric-assay/figure-c1.png)

Figure C1. The agarose gel electrophoresis result of insert digestion. Lane 1: 100 bp DNA
ladder. Lane 2: Insert\_0004771 digestion product. Lane 3: Insert\_0101802 digestion product.

# Analysis

Lane 2: According to the DNA ladder, the base pair of digested
Insert\_0004771is between
200 bp and 300 bp, which conforms to our expectations.

Lane 3: According to the DNA ladder, the base pair of digested
Insert\_0101802 is
between 300 bp and 400 bp, which conforms to our expectations.

# B. Insert ligation

# Data

![](https://static.igem.wiki/teams/4636/wiki/results-colorimetric-assay/figure-c2.png)

Figure C2. The agarose gel electrophoresis result of insert ligation. Lane 1: 1 kb DNA ladder,
Lane 2: Insert\_0004771 ligation product, Lane 3: Insert\_0101802 ligation product.

# Analysis

Lane 2: According to above figure, we discovered that the insert sequences are ligated in
different fold numbers. Therefore, the result of agarose gel electrophoresis showed multiple
bands with equal interval in Lane 2.

Lane 3: Similar to Lane 2, it also showed multiple bands with equal interval in Lane 3. The
difference between the two lanes was that the band position of Insert\_0101802 (Lane 3) was
higher than Insert\_0004771 (Lane 2).

# Achievement

We successfully ligated each single piece of insert into a long-repeated dsDNA.

# Preparation of template for AELA-PCR

Description: We reintroduced the cloning process in an attempt to incorporate additional
sequences and amplify the long-repeated insert sequences

# A. Colony PCR

# Data

![](https://static.igem.wiki/teams/4636/wiki/results-colorimetric-assay/figure-c3.png)

Figure C3. The agarose gel electrophoresis result of colony PCR. Lane 1: 1kb DNA ladder, Lane 2
to Lane 5: the samples of colony PCR were picked from four different colonies.

# Analysis

We observed the absence of bands in Lane 2, 4 and 5. In
Lane 3, although there was a band present, its length fell between 500-1000 bp, shorter than our
long-repeated insert sequence (approximately 1680 bp in total, 336 bp per piece). In summary, we
did not achieve the expected amplification of the inserts.

# B. Plasmid gel electrophoresis

# Data

![](https://static.igem.wiki/teams/4636/wiki/results-colorimetric-assay/figure-c4.png)

Figure C4. The agarose gel electrophoresis result of plasmid. Lane 1: 1 kb DNA ladder. Lane 2:
pUC19 vector only. Lane 3: pUC19 vector ligated with long-repeated insert sequence.

# Analysis

Lane 2: Only the pUC19 vector was loaded into Lane 2. The vector is around 2686 bp long.

Lane 3: We ligated the pUC19 vector (which is 2686 bp) with a long-repeated insert sequence
(1680 bp in total, 336 bp per piece). The band marked with a red frame was around 4366 bp
(vector 2686 bp + insert 1680 bp), which indicated the successful ligation.

# Achievement

We successfully ligated each single piece of insert into a long-repeated dsDNA.

# AELA PCR

Since we failed to amplified the long-repeated insert sequences utilizing colony PCR as
mentioned before. We could not further execute the experiments afterwards.

# Path D: Amplify CircRNA by RCA for Colorimetric Assay Detection

**Sequence design → Cloning → PCR → IVT → Circularization → CircRNA enrichment → RCA → Probe
conjugated with gold nanoparticles (AuNPs) detection**

# Rolling Circle Amplification (RCA)

# Description

Rolling Circle Amplification (RCA) is a kind of isothermal amplification method that can
generate long-repeated complementary DNA (cDNA) of circRNA by reverse transcriptase. Compared to
circular form RNA, linear RNA can only be reverse transcribed into complementary DNA (cDNA) with
a length identical to that of the RNA template. Thus, in this experiment, we compared the
amplification of linear form and circular form RNAs, to confirm whether RCA can generate
long-repeated complementary DNA (cDNA) longer than the circRNA.

# Data

![](https://static.igem.wiki/teams/4636/wiki/results-colorimetric-assay/figure-d1.png)

Figure D1. Gel electrophoresis results verify the RCA process. Lane 1: 1000 bp ssDNA ladder,
Lane 2: Circular RNA (202 ng) after 15 minutes RCA amplification, Lane 3: Circular RNA (202 ng)
after 30 minutes RCA amplification, Lane 4: Low concentration of linear RNA (202 ng) after RCA
15 minutes amplification, Lane 5: Low concentration of linear RNA (202 ng) after RCA 30 minutes
amplification, Lane 6: High concentration of linear RNA (2457.2 ng) after RCA 15 minutes
amplification, Lane 7: High concentration of linear RNA (2457.2 ng) after RCA 30 minutes
amplification.

# Analysis

In Figure D1, compared Lane 2 with Lane 4, we found that
Lane 2 showed a higher position than
Lane 4, indicating the successful generation of long-repeated complementary DNA (cDNA). By
comparing Lane 4 to Lane 5, we concluded that conducting RCA for different time intervals does
not significantly affect the results. Additionally, comparing Lane 7 to Lane 5, we observed that
a higher sample concentration leads to a greater yield of reverse transcription products.

# Achievement

Successfully generated a repeated cDNA sequence which is
longer than the circRNA sequence for use in the gold nanoparticles colorimetric assay.

# Probe conjugated with gold nanoparticles (AuNPs) detection

# Description

To determine whether a patient is infected with the
disease, we can visually observe the color change of the gold nanoparticle solution under
different conditions. According to previous studies, the addition of MgCl2 to the AuNPs solution
induces aggregation and a color change (to purple). However, if the probes conjugated on the
gold nanoparticles recognize and bind to the RCA product, the long-repeated cDNA sequence
prevents AuNPs aggregation and also the color change (red) when MgCl2 is added.

Unfortunately, due to time constraints, we were unable to
generate a sufficient amount of long-repeated cDNA for testing with Probe conjugated with gold
nanoparticles (AuNPs) detection. Therefore, in this section, we are initially adding RCA NTC
solution to Probe-Conjugated Gold Nanoparticles and testing the relationship between the volume
of 0.21 M MgCl2 added and the resulting color change. In the future, if we have enough
long-repeated cDNA, we will be able to test whether probe-conjugated gold nanoparticles can
recognize and bind to it, thereby maintaining the color.

# Data

![](https://static.igem.wiki/teams/4636/wiki/results-colorimetric-assay/figure-d2.png)

Figure D2. The spectrum of adding MgCl2 to Probe conjugated-AuNP containing RCA solution.

![](https://static.igem.wiki/teams/4636/wiki/results-colorimetric-assay/figure-d3.jpg)

Figure D3. The color of the Probe-Conjugated Gold Nanoparticles solution is influenced by the
varying volumes of added 0.21 M MgCl2.

# Analysis

In Figure D2. we can observed a red shift in spectrum after
adding MgCl2 to the Probe conjugated-AuNP with no target cDNA. Besides, in Figure D3, a
significant color change can be observed when adding 6-8 µL 0.21M MgCl2.

# Achievement

We find out that 6–8 µL 0.21 M MgCl2 added to the
Probe-Conjugated Gold Nanoparticles solution causes the color change and a redshift in spectrum.

# Path E: Access the Functionality of Lateral Flow Test by RPA Products

**Sequence design → Cloning → PCR → RPA → PCRD**

# Recombinase Polymerase Amplification (RPA)

# Description

Before RT-RPA, the ultimate amplification method of our
choice, we adopted only RPA to confirm that the RPA primers we designed can work properly. After
the reaction, gel electrophoresis was performed to check whether the band positions of the
amplicons represented the same length as our inserts.

# Data

![](https://static.igem.wiki/teams/4636/wiki/results-lateral-flow-test/figure-e1.png)

Figure E1. RPA 2% agarose gel electrophoresis results. Lane 1: Bio-100 Mass DNA Ladder, Lane 2:
Insert\_0004771+primer 1 (RPA\_0004771fw/rv\_1), Lane 3 : NTC of Lane 2, Lane 4:
Insert\_0004771+primer 2 (RPA\_0004771fw/rv\_2), Lane 5: NTC of Lane 4, Lane 6: X , Lane 7:
Insert\_0101802+primer 1 (RPA\_0101802fw/rv\_1), Lane 8 : NTC of Lane 7, Lane 9:
Insert\_0101802+primer 2 (RPA\_0101802fw/rv\_2), Lane 10 : NTC of Lane 9.

# Analysis

We confirmed the successful amplification of our target circRNA after RPA by verifying the
length of the amplicons. However, the electrophoresis result showed that in RPA reaction of
Insert\_0004771, bands were observed in no template control (NTC) groups, futhermore, the control
group and the sample-treated group had the same band positions. We suspect that the reagents
might have been contaminated by the sample, leading to this result.

# Achievement

The RPA primer we designed can function as expected, hence we could get into the next path—using
linear RNA, which is the product of in vitro transcription (IVT), to conduct RT (reverse
transcription)-RPA.

# PCRD

We conducted PCRD lateral flow test using amplicons
produced from RT-RPA. (See Path F. PCRD)

# Path F: Conducting RT-RPA Using Linear RNA as template for Lateral Flow Test

**Sequence design → Cloning → PCR → IVT → RT-RPA → PCRD**

# RT-RPA

# A. RT-RPA Analysis of Serially Diluted Linear RNA Samples

# Description

Since we have ensured that the primers we designed can
successfully work in RPA, we conducted a reverse transcription (RT)-RPA to amplify the RNA,
which was obtained from in vitro transcription (IVT) of our insert (cDNA) (See Results—CircRNA
Synthesis).

# Data

![](https://static.igem.wiki/teams/4636/wiki/results-lateral-flow-test/figure-f1.jpg)

Figure F1. Perform RT-RPA on a serial dilution of linear RNA samples. Lane 1: No Template
Control (NTC); Lane 2: RT-RPA using IVT product of Insert\_0004771 (68.2 ng/μL); Lane 3: A
10^2-fold dilution of the IVT product from Insert\_0004771 (68.2 ng/μL); Lane 4: A 10^4-fold
dilution of the IVT product from Insert\_0004771 (68.2 ng/μL); Lane 5: A 10^6-fold dilution of
the IVT product from Insert\_0004771 (68.2 ng/μL); Lane 6: 100 bp DNA Ladder; Lane 7: No Template
Control (NTC); Lane 8: RT-RPA of the IVT product from Insert\_0101802 (97.8 ng/μL); Lane 9: A
10^2-fold dilution of the IVT product from Insert\_0101802 (97.8 ng/μL); Lane 10: A 10^4-fold
dilution of the IVT product from Insert\_0101802 (97.8 ng/μL); Lane 11: A 10^6-fold dilution of
the IVT product from Insert\_0101802 (97.8 ng/μL).

# Analysis

It can be confirmed that reverse transcriptase could effectively work together with RPA in a
single step, as indicated by the clear and light band observed in our RT-RPA results.

Additionally, both of the no template control (NTC) using different pair of forward and reverse
primers showed bright and clear band. The exist of band in (NTC) may caused by following
reason:
(1) The contamination of DNA samples in RT-RPA reagents.
(2) The formation of self primer-dimer.
(3) The formation of hetero primer-dimer between forward and reverse primer.

# B. PCR Analysis of RT-RPA Reagents for DNA samples Contamination

# Description

Since we observed a band in the no template control (NTC)
lane of RPA and RT-RPA experiment during gel electrophoresis, we suspected that contamination of
the samples ( Insert\_0004771 and Insert\_0101802) may have contributed to this result. To confirm
whether the reagents contained sample contamination, we conducted PCR tests on all RT-RPA
reagents, including primers. The PCR tests were performed by utilizing PCR\_0101802fw and
PCR\_0101802rv primers.

# Data

![](https://static.igem.wiki/teams/4636/wiki/results-lateral-flow-test/figure-f2.jpg)

Figure F2. Performance of PCR on the RT-RPA reagents to test for contamination. Lane 1: 100 bp
DNA Ladder; Lane 2: Primer free rehydration buffer; Lane 3: RPA\_0004771fw\_1; Lane 4:
RPA\_0004771rv\_1; Lane 5: RPA\_0004771fw\_2; Lane 6: RPA\_0004771rv\_2; Lane 7: RNase Inhibitor; Lane
8: dNTP; Lane 9: Magnesium Acetate (MgOAc); Lane 10: Nuclease-free water; Lane 11:
ProtoScript®II Reverse Transcriptase.

# Analysis

By conducting PCR tests on all RT-RPA reagents, the results revealed that bands occurred in Lane
2, Lane 4, and Lane 6, corresponding to the reaction using primer-free rehydration buffer,
forward primer, or a reverse primer as a sample, respectively. These results indicate the
primer-free rehydration buffer and the primers may have been contaminated.

# C. RT-RPA Self-Primer Dimer Testing

# Description

Since we observed a band in the no template control (NTC)
lane of the RT-RPA experiment during gel electrophoresis, we suspected that the formation of
self-dimer may have caused this result. Therefore, in this experiment, our objective is to add
only one primer at once to assess the possibility of self-primer dimer formation during reaction
of (NTC).

# Data

![](https://static.igem.wiki/teams/4636/wiki/results-lateral-flow-test/figure-f3.jpg)

Figure F3. Test of Self Primer Dimer. Lane 1: 100 bp DNA Ladder; Lane 2:
RPA\_0004771fw\_1+RPA\_0004771rv\_1; Lane 3: RPA\_0004771fw\_2+RPA\_0004771rv\_2; Lane 4:
RPA\_0004771fw\_1 only; Lane 5: RPA\_0004771rv\_1 only; Lane 6: RPA\_0004771fw\_2 only; Lane 7:
RPA\_0004771rv\_2 only.

# Analysis

Lane 4, Lane 5, Lane 6, and Lane 7 showed a faint band in the RT-RPA gel electrophoresis result,
this may indicate the possibility of self-dimer formation. However, compared to Lane 1 and Lane
2, the positions of Lane 4, Lane 5, Lane 6, and Lane 7 are lower. Hence, it is less likely that
the prominent band observed in the no template control (NTC) lane of the RT-RPA experiment is a
primer's self-dimer.

# D. RT-RPA Hetero-Primer Dimer Testing through NTC Sequencing

# Description

To determine whether the band observed in the no template
control (NTC) lane of the RT-RPA experiment is a result of sample contamination or the formation
of hetero-dimer between forward and reverse primer, we conducted a repeat (NTC) of RT-RPA using
forward primer RPA\_0004771fw\_1 and reverse primer RPA\_0004771rv\_1. Subsequently, we submitted
the sample for Sanger sequencing at Genomics (a commercial genome sequencing company in Taiwan)
and align the resulting sequence with our insert sequence by NCBI sequence alignment tool.

# Data

![](https://static.igem.wiki/teams/4636/wiki/results-lateral-flow-test/figure-f4-sequencing-the-ntc-band.jpg)

Figure F4. Negative Template Control (NTC) of RT-RPA, using RPA\_0004771fw\_1 and RPA\_0004771rv\_1.

![](https://static.igem.wiki/teams/4636/wiki/results-lateral-flow-test/figure-f5.png)

Figure F5. Alignment of the Sanger sequencing result with the sequence theory amplified by
RPA\_0004771fw\_1 and RPA\_0004771rv\_1 primers, which corresponds to Insert\_0004771.

# Analysis

The result from Sanger sequencing (Figure F5.) indicated a 99% similarity to our target RNA
sequence. We have concluded that the band observed in the no template control (NTC) lane is the
result of contamination. Therefore, we ordered new primers and primer-free rehydration buffer
for further experiments.

# E. RT-RPA New Primer Testing

# Description

Since the band in the no template control (NTC) lane of the
RT-RPA experiment during gel electrophoresis is caused by contamination, we used new primers and
primer-free rehydration buffer in this experiment.

# Data

![](https://static.igem.wiki/teams/4636/wiki/results-lateral-flow-test/figure-f6.jpg)

Figure F6. NTC of RT-RPA using different primers. Lane 1: 100 bp DNA Ladder; Lane 2: old
RPA\_0004771fw\_2+RPA\_0004771rv\_2; Lane 3: new RPA\_0004771fw\_1+RPA\_0004771rv\_1; Lane 4: new
RPA\_0004771fw\_2+RPA\_0004771rv\_2.

# Analysis

Comparing lane 4 with lane 2, there was no band presented in the no template control (NTC) of
RT-RPA using the new rehydration buffer and primers. This indicates that the contamination is
coming from the rehydration buffer and primers.

# F. Determining the Amplification Threshold of RT-RPA

# Description

After successfully conducting the no template control (NTC)
of RT-RPA, we can now proceed with further experiments. We have diluted our linear RNA sample
(generated by IVT) and determined the limit of amplification using RT-RPA. Besides, in this
experiment, we added 1.5 μL of both forward and reverse primers (10 μM each) and allowed them to
react for 10 minutes.

# Data

![](https://static.igem.wiki/teams/4636/wiki/results-lateral-flow-test/figure-f7-gel-electrophoresis-of-diluted-rna-sample-from-rt-rpa.jpg)

Figure F7. Gel electrophoresis of diluted RNA sample from RT-RPA. Lane 1: 100 bp DNA Ladder,
Lane 2: RT-RPA using IVT product of circ\_0101802 (385 ng/μL), Lane 3: A 10^4-fold dilution of
the IVT product from Insert\_0101802 (385 ng/μL); Lane 4: A 10^6-fold dilution of the IVT product
from Insert\_0101802 (385 ng/μL), Lane 5: A 10^8-fold dilution of the IVT product from
Insert\_0101802 (385 ng/μL), Lane 6: A 10^10-fold dilution of the IVT product from Insert\_0101802
(385 ng/μL), Lane 7: A 10^12-fold dilution of the IVT product from Insert\_0101802 (385 ng/μL),
Lane 8: No template control (NTC).

# Analysis

As we seen in the gel electrophoresis results, the lower concentration of RNA sample, the
lighter its band displays. Additionally, we can found out that the band is very light at
diluting the sample solution to 10^-12 times (approximately 3.52\*10^-10 ng/μL), thus we
speculate that the limit of sample amplification for RT-RPA should be around 3.52\*10^-10 ng/μL
when we added 1.5 μL of both forward and reverse primer (10 μM) and react for 10 minutes.

# G. Amplify circRNA by RT-RPA

# Description

After successfully amplifying linear form RNA through RT-RPA, we switched our samples from linear form RNA to circular form RNA for RT-RPA.

# Data

![](https://static.igem.wiki/teams/4636/wiki/results-lateral-flow-test/20231021rt-rpa.jpg)

Figure F8. The gel electrophoresis results confirmed the success of the RT-RPA process for circRNA. Lane 1: 100 bp DNA Ladder, Lane 2: No Template Control, Lane 3: Linear form RNA, Lane 4: Circular form RNA

# Analysis

As we seen in the gel electrophoresis results (Figure F8), both lane 3 and lane 4 have band at the same position, indicate we successfully amplify circRNA through RT-RPA.

# PCRD

# Description

Our outcomes indicated that unnecessary oligonucleotides might form during RT-RPA reactions due
to primer-dimers, which remained an unsolved problem. Despite that, we could still conduct
lateral flow test with PCRD to confirm whether the customized lateral test strip we designed
possessed the ability to catch the labeled-amplicons on it.

Lateral flow test was performed to detect RPA amplicons labeled with digoxigenin (DIG)/biotin
and carboxyfluorescein (FAM)/biotin. PCRD cassette (Abington Health) (Figure F8) was used to
visualize DIG/biotin or FAM/biotin-labeled amplicons.

| RT-RPA sample (See Parts\_Composite parts for more info) | 5' modification of primer (See Sequence Design—RPA Primer to learn about primer design) | RT-RPA product | Corresponding line on the lateral flow strip | Antibody type |
| --- | --- | --- | --- | --- |
| Insert\_0004771 | Forward primer: biotin Reverse primer: DIG | DIG/biotin labeled-amplicons | Test line 1 | Anti-DIG capture antibody |
| Insert\_0101802 | Forward primer: biotin Reverse primer: FAM | FAM/biotin labeled-amplicons | Test line 2 | Anti-FAM capture antibody |
| / | / | / | Control line (C) | Anti-mouse antibody |

![](https://static.igem.wiki/teams/4636/wiki/results-lateral-flow-test/fig-f8.jpg)

Fig F8. Composition of a PCRD lateral flow cassette. When the sample is applied to the cassette,
the buffer flows down the strip, carrying the carbon-conjugated biotin antibodies and the
amplicons with it. If the amplicons are present in the sample, they will bind to the anti-DIG
monoclonal antibodies on the test line 1, or anti-FAM antibodies on the test line 2. The carbon
particles will accumulate on the test line, making it visible. The rest of carbon-conjugated
biotin antibodies will be captured on the C line, which can be served as control.

We utilized the product of RT-RPA directly to conduct
lateral flow test. Three kinds of RNA samples were used in RT-RPA reaction: A. Insert\_0004771
and Insert\_0101802 mix, B. Insert\_0101802 only, and C. No template control (NTC).

# Data

![](https://static.igem.wiki/teams/4636/wiki/results-lateral-flow-test/figure-f9.jpg)

Fig F9. Results of RT-RPA amplicons identification after 20 min incubation at 39 °C using a
PCRD cassette: (A) RT-RPA amplicons of in-vitro transcription (IVT) products of Insert\_0004771
and Insert\_0101802 were added, two black test lines and a control line occured, revealing the
successful binding of labeled-amplicons and antibodies on the strip, (B) The second test line
corresponded with the RT-RPA product using in-vitro transcribed RNA of Insert\_0101802 only, and
(C) Only the control line presents when no template control (NTC) is added.

# Analysis

In theory, there should be only a control line presented on
the lateral flow strip when no target sequences are added (no template control), as shown in
Figure F9. Nevertheless, our no template control (NTC) results showed a test line during the
lateral flow test. To address this unexpected outcome, we learned from a previous study [[3]](#[3]) and
concluded that the formation of primer-dimers by the modified reverse and forward primers can
lead to false-positive results. Therefore, we plan to design more specific primers in the
future.

Our experimental results confirm that when target RNA is
present in the RT-RPA reaction, it can be successfully amplified and show a positive result when
further tested by PCRD. This observation is consistent with the expected outcome of our assay.

# Achievement

We confirmed that the modified RPA primer can work through
RT-RPA reaction, and made sure the reverse transcriptase works by conducting RT-RPA reaction. At
last, confirming the modified primers we designed can generate labeled amplicons and
successfully bind with corresponding antibodies. This was observed by examining the test lines
on PCRD.

# Path G: Amplify CircRNA Utilizing RT-RPA for Lateral Flow Test

Unfortunately, due to time constraints, we were unable to complete this part before Wiki freeze.
A significant portion of our time was devoted to determining the amplification threshold of
RT-RPA, optimizing reaction times, primer concentrations, and addressing contamination issues.
Consequently, amplify circRNAs utilizing RT-RPA will be a key focus part for our future work.

# Reference

[1] Ali, M. M., Kanda, P., Aguirre, S. D., & Li, Y. (2011). Modulation of DNA-modified
gold-nanoparticle stability in salt with concatemeric single-stranded dnas for colorimetric
bioassay development. **Chemistry - A European Journal**, **17**(7), 2052–2056.
<https://doi.org/10.1002/chem.201002677>

[2] Li, F., Zhang, H., Dever, B., Li, X.-F., & Le, X. C. (2013). Thermal stability of DNA
functionalized gold nanoparticles. **Bioconjugate Chemistry**, **24**(11), 1790–1797.
<https://doi.org/10.1021/bc300687z>

[3] Li, C.-J., Sun, H.-Q., Zhao, W.-X., Wang, X.-Y., Lin, R.-Z., & Yao, Y.-X. (2023). Rapid
Assay Using Recombinase Polymerase Amplification and Lateral Flow Dipstick for Identifying
Agrilus Mali, a Serious Wood-Boring Beetle of the Western Tianshan Mountains in China.

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