iGEM 2022 — Team tsinghua

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
{% block page\_content %}

# NOTEBOOK

## Click anywhere to open

Dec.

Jan.

Feb.

Mar.

Apr.

May

Jun.

Jul.

Aug.

Sep.

Oct.

## December

Week 51

Week 52

# Week of December 20th to 26th

The previous team members were worked through two days of interviews and 16 members of Tsinghua igem 2022 were selected! Relay baton is now in our hands!

# Week of December 27th to January 2nd

Tsinghua iGEM 2022 had its first meeting and it was indeed awkward, maybe we didn't recognize each other clearly.
![](https://static.igem.wiki/teams/4307/wiki/wikinotebookfiles/preface/1.jpg)

![](https://static.igem.wiki/teams/4307/wiki/wikinotebookfiles/preface/2.jpg)

## January

Week 1

Week 2

Week 3

Week 4

Week 5

# Week of January 3rd to 9th

Winter break started and we had our first meeting offline. The Tsinghua iGEM 2021 leaders explained the arrangements for winter break and everyone was still very ignorant about iGEM.
![](https://static.igem.wiki/teams/4307/wiki/wikinotebookfiles/preface/3.png)

# Week of January 10th to 16th

In order to grasp the rules of the iGEM competition initially，our assignment for the first week was to select an iGEM project from previous years for research and study, and to complete a 20min presentation in groups of four.
![](https://static.igem.wiki/teams/4307/wiki/wikinotebookfiles/preface/4.png)

# Week of January 17th to 23th

The second week's assignment was to regroup and choose a track conduct research.
![](https://static.igem.wiki/teams/4307/wiki/wikinotebookfiles/preface/5.png)

# Week of January 24th to 30th

This week's assignment was to do research on special awards, and a dozen special awards were divided into four topics. Still regrouping.
![](https://static.igem.wiki/teams/4307/wiki/wikinotebookfiles/preface/6.png)

# Week of January 31th to February 6th

This week's assignment is to read the iGEM 2021 judging book, which is the last assignment for our winter break!
![](https://static.igem.wiki/teams/4307/wiki/wikinotebookfiles/preface/7.png)
Since we found out in last week's presentation that the education part can be done long before the topic of the project is determined, we formed a group this week to work on the science education videos.

## February

Week 6

Week 7-8

Week 9

# Week of February 7th to 13th

During these two weeks, we divided into four groups for brainstorm and presented the projects we wanted to work on in iGEM this year. This is the checkpoint of our brainstorm 1.
![](https://static.igem.wiki/teams/4307/wiki/wikinotebookfiles/preface/8.jpg)

# Week of February 14th to 20th and Week of February 21st to 27th

This week was checkpoint 2, where we refined and improved the projects presented last week based on the suggestions from other team members in the meeting. We each scored the projects of each group.
![](https://static.igem.wiki/teams/4307/wiki/wikinotebookfiles/preface/9.jpg)

# Week of February 28th to March 6th

We regrouped and started the brainstorm for the second round.
![](https://static.igem.wiki/teams/4307/wiki/wikinotebookfiles/preface/10.png)

## March

Week 10-11

Week 12

Week 13

# Week of March 7th to 13th and Week of March 14th to 20th

We regrouped and started the brainstorm for the second round.
![](https://static.igem.wiki/teams/4307/wiki/wikinotebookfiles/preface/10.png)

# Week of March 21st to 27th

As with the first round of brainstorm, this week was a time of iteration for the project.
After the presentation, we scored again. The scores of the eight ideas from the two brainstorm rounds were sorted together and we will choose the two groups with the highest scores for pre-experimentation.
![](https://static.igem.wiki/teams/4307/wiki/wikinotebookfiles/preface/11.jpg)

Although other ideas were not chosen, we are still impressed by the creativity behind these ideas.

# Week of March 28th to April 3rd

It was the first week of the pre-lab, and each of our two groups presented their plans for the next month-long pre-experiments at meeting. Although there would be no more battles or choices to make this week, everybody was still very nervous.

## April

Week 14

Week 15

Week 16

Week 17

# Week of April 4th to 10th

This is the time we got to our midterm week, everyone was a bit busy and there was no group meeting this week.

# Week of April 11th to 17th

Affibody-Pmr: the fusion protein sequence has been designed and we have ordered it in the gene synthesis platform.
Nisin: we tried to obtain the sequence of nisA by pcr with short primers, but failed and it seems that we still need to purchases it.
We obtained Lactobacillus from the previous igem teams and performed colony PCR of 16SrRNA to verify.We had our new member in iGEM 2022: a pillow that is indeed so long. we call it worm dog.

# Week of April 18th to 24th

Affibody-Pmr: We obtained EGFR fragments from plasmids in iGEM 2021.
Nisin: We asked iGEM 2021 players how we should choose vectors and identified pET28a and pET32a as the governing vectors and started to design a cloning strategy.

# Week of April 25th to May 1st

We're on Labor Day holiday this week, not much progress, and we had a big clean-up, let's all get some rest!

## May

Week 18

Week 19

Week 20

Week 21

Week 22

# Week of May 2nd to 8th

 Nisin: This week we finished the construction and transformation of the experimental plasmids.
Also, during the experiment we realized that Hifi is efficient as a means to construct clones, and the protocol and system of the experiment are gradually fixed and standardized.

# Week of May 9th to 15th

This week we had our final presentation of two projects and we are gonna choose the projects we will be working on in iGEM2022 by secret ballot.
Before the presentation took place, we had a captain's election.
Since it was a life and death week, our experiments were very heartfelt. Our biggest concern: whether or not the two-component system would work in E. coli, finally came to fruition this week. At this point we were still very new to the induction of the two-component system, and we were just starting to experiment with the induction time, the amount of induce molecule, and the incubation temperature. Also, fluorescence was not as easy to observe as we previously thought, and team members in iGEM 2021 taught us how to use the fluorescence microscope. We still remember the night when we were in the fluorescence microscope classroom observing the fluorescent bacteria with great anxiety, but the light of the slow flowing fluorescent bacteria greatly soothed our mood.
Thanks to our positive results, the sperm selection finally won in the pre-experiment stage!

# Week of May 16th to 22th

As we approached the end of the semester, we divided our team members into groups , arranged the tasks that needed to be completed before the beginning of the summer.

# Week of May 23th to 29th

Unfortunately we are going to be in quarantine at school, and we have to decide whether we are gonna stay in school or go home. it is about how many people there will be here for wetlab in summer break. We had a vote.
On the meeting this week, our PI Li Peng had us team training. We are touched and felt even closer to each other.

# Week of May 30th to June 5th

And we held a picnic in our campus. We love pizza.

## June

Week 23

Week 24

Week 25

Week 26

# Week of June 6th to 12th

One of our leader in iGEM2021 is going to leave school and studying abroad. We gathered together to take picture with him. (Love you World!)

# Week of June 13th to 19th

This is the first week of our summer vacation, before this week started; we had a summer pep rally to sort out our progress in wetlab, drylab and HP, and put forward our plans in the summer; we invited the leader of Tsinghua iGEM 2021 to share our experience on how to plan our summer vacation; finally, each of our team members shared their vision when first joined in iGEM 2022 , hoping to keep our project improving during the summer.
We wrote our Impact Grant application this week and invited our Tsinghua iGEM 2019 team captain, Huang Zikang, to give us some advices and hopes that we would be successful in winning the award! (And it did!)
In terms of lab management, we organized the -20° refrigerator, so we finally don't have to rummage through boxes to find things; at the same time, we standardized the format of experiment records on benchling, and pre-saved several templates for common experiments, so perhaps it will be more convenient to record experiments.

# Week of June 20th to 26th

## Pmr and Amplifier

### Week Plan:

1. Plasmid construction of pET28a-AffiPmrB-aa.
2. Plasmid construction of pET28a-AffiPmrB-NEU.
3. Preliminary verification of AffiPmrB-A-C system.

### Progress:

1. Plasmid construction of pET28a-AffiPmrB-aa.
We prepared PCR fragments for HiFi assembly.
2. Plasmid construction of pET28a-AffiPmrB-NEU.
We successfully constructed this plasmid.
3. Preliminary verification of AffiPmrB-A-C system.
We did verification by fluorescence microscope.

### Troubleshooting:

1. Plasmid construction of pET28a-AffiPmrB-aa.
2. Plasmid construction of pET28a-AffiPmrB-NEU.
3. Preliminary verification of AffiPmrB-A-C system.
Problem:
The fluorescence intensity was too low.
Solution:
We plan to change our induction protocol and use other methods for verification.

### Future Plan:

1. Plasmid construction of pET28a-AffiPmrB-aa.
2. Verification of AffiPmrB-A-C system.

## Nisin

### Week Plan:

1. Plasmid construction of pNisAB and pNisAB(-)
which contains NisA instead of NisA-affi
2. Plasmid construction of pNisC
3. Plasmid construction of pNisinOE2

### Progress:

1. Plasmid construction of pNisAB and pNisAB(-):
We have done the Hifi assembly.
2. Plasmid construction of pNisC:
We successfully constructed this plasmid.
3. Plasmid construction of pNisinOE2:
We successfully constructed this plasmid.

### Future Plan:

1. Complete plasmid construction of pNisAB and pNisAB(-)
2. Cotransform the pNisAB and pNisC to BL21(DE3)
3. Verify the expression of NisA-affi
4. Transform the pNisinOE2 to BL21(DE3)
5. Induce lacZ expression of pNisinOE2 with 1ng/ml nisin

## Logic Gate

### Week Plan:

1. Plasmid construction of v13. pET28a-ser integrase
2. Plasmid construction of v19. pUC-mCherry-attB-OR-attP-EGFP

### Progress:

1. Plasmid construction of v13. pET28a-ser integrase:
We failed to construct this plasmid.
2. Plasmid construction of v19. pUC-mCherry-attB-OR-attP-EGFP:
We failed to insert mCherry fragment into the pUC vector containing the recombination sites of serine integrase.
![](https://static.igem.wiki/teams/4307/wiki/wikinotebookfiles/logicgate/1.png)

### Troubleshooting:

1. Cro&cI function verification:
Problem:
Under the optimal induction condition, cI and cro both seemed to function. However, cro seem to be less effective than the positive control.
Analysis:
Conclusions were too soon to be drawn before we repeated the experiment for several times.
Solution:
We planned to repeat this experiment.
2. qPCR result:
Problem:
No significant result can be seen for qPCR even though microplate reading indicated stark contrast of fluorescence intensity before and after the induction. Several wells even showed no ct after qPCR, yet correct bands can be seen after we ran the qPCR product on the gel.
Analysis:
We speculated that our qPCR system was somewhat inappropriate.
Solution:
We planned to optimize our qPCR protocol and system.

### Future Plan:

1. Cro&cI function verification: microplate reading to test new cro
2. Serine integrase function verification: qPCR

## HP

It was the first week of the summer vacation and we were about to start our HP. We contacted Ms. Lan Xie, whom we had consulted when we first decided the topic. She was teaching a course on reproductive health at Tsinghua University and had done some research on sperm quality test. At this time, we did not know much about current hospital sperm test and were still looking for a breakthrough point that we could target. Through our conversation with her, we learned about the current sperm testing methods in the hospital. She also provided constructive comments on our project.

## Others

This week, our artwork girl sorted out the painting tasks expected to be completed during the summer holidays.
![](https://static.igem.wiki/teams/4307/wiki/wikinotebookfiles/others/0.png)
(The comic she drew was so cute!!)
The preliminary design of the Modeling section was also started this week, giving the output of the fluorescent protein under different starting protein concentration conditions.

# Week of June 27th to July 1st

## Pmr and Amplifier

### Week Plan:

1. Plasmid construction of pET28a-AffiPmrB-aa.
2. Verification of AffiPmrB-A-C system.

### Progress:

1. Plasmid construction of pET28a-AffiPmrB-aa.
We failed to construct the plasmid.
2. Verification of AffiPmrB-A-C system.
We did verification of the system by confocal.

### Troubleshooting:

1. Plasmid construction of pET28a-AffiPmrB-aa.
Problem:
The one-step-clone showed positive results by PCR, but sequencing showed negative results instead.
Analysis:
We found that the PCR primers we used to do PCR tests were somehow contaminated by template DNA, therefore the PCR couldn't screen out the positive clone.
Solution:
We changed all our primers and fragments for clone.
2. Verification of AffiPmrB-A-C system.

## Nisin

### Week Plan:

1. Complete plasmid construction of pNisAB and pNisAB(-)
2. Cotransform the pNisAB and pNisC to BL21(DE3)
3. Verify the expression of NisA-affi
4. Transform the pNisinOE2 to BL21(DE3)
5. Induce lacZ expression of pNisinOE2 with 1ng/ml nisin

### Progress:

1. Complete plasmid construction of pNisAB and pNisAB(-):
We successfully constructed this plasmid.
2. Cotransform the pNisAB and pNisC to BL21(DE3)
The transformation is successful, but the efficiency is very low. Only one colony grew in the two plates.
3. Verify the expression of NisA-affi
Be limited by cotransformation efficiency, the work didn't begin this week.
4. Transform the pNisinOE2 to BL21(DE3)
We successfully tranform the plasmid to BL21(DE3).
5. Induce lacZ expression of pNisinOE2 with 1ng/ml nisin
The colonies were all blues whether it was induced by nisin or not.
![](https://static.igem.wiki/teams/4307/wiki/wikinotebookfiles/nisin/week1.jpg)

### Troubleshooting:

1. Low transformation efficiency
Problem:
The transformation is successful, but the efficiency is very low. Only one colony grew in the two plates.
Analysis:
Maybe the concentration of antibiotics in the solid medium is too high which inhibits the bacteria to grow.
Solution:
We planned to reduce the concentration of anbibiotics in the solid medium. We also planned to do cotransformation with electric-transformation method.
2. Failure of inducing expression of lacZ
Problem:
The colonies were all blues whether it was induced by nisin or not.
Analysis:
BL21(DE3) has endogenous lacZ expression.
Solution:
We planned to knock out the endogenous lacZ gene of BL21(DE3) or find out E.coli strain which has not endogenous lacZ gene.

### Future Plan:

1. Cotransformation of pNisAB and pNisC: continue
2. Verify the expression of NisA-affi

## Logic Gate

### Week Plan:

1. Plasmid construction of v13. pET28a-ser integrase
2. Plasmid construction of v19. pUC-mCherry-attB-OR-attP-EGFP

### Progress:

1. Plasmid construction of v13. pET28a-ser integrase:
We successfully constructed this plasmid.
2. Plasmid construction of v19.
pUC-mCherry-attB-OR-attP-EGFP
We successfully inserted EGFP fragment into pUC vector containing the recombination sites of serine integrase, but still failed to insert mCherry fragment.
![](https://static.igem.wiki/teams/4307/wiki/wikinotebookfiles/logicgate/2.png)

### Troubleshooting:

Problem:
The PCR result of linearization always showed ladder-like bands, resulting in low concentration after gel extraction.
Analysis:
We speculated unspecific binding of the primers to the template.
Solution:
We planned to redesign the primers to enhance specificity in the hope of a better linearization result.
Future Plan:
1. Plasmid construction of v19.
pUC-mCherry-attB-OR-attP-EGFP: continue
2. Serine integrase function verification: preliminary experiment

## HP

This week we reached out to Dr. Yiqun Gu of the National Sperm Bank, a researcher studying male reproductive health. Two of our members met with him face-to-face. During our conversation with him, we learned more about the current hospital sperm test and the drawbacks of the current home sperm test strips. He recognized the innovative nature of the parts used in our project, but he suggested that our project still needed to consider practicality and the need to reduce costs as well as be user-friendly.

## Education

This week, we had the idea of joining forces with teams from iGEM teams in China for a joint science education project during an exchange with JLU and ShangTech students in charge of education.

## Others

We revisited the entire project design this week and realized that the current design has the problem of long detection times. We discussed at the group meeting how to make changes to the project, even including reversing the entire project. The conclusion was to spend a little time in the following week brainstorming in groups and presenting at the group meeting the following week.

## July

Week 27

Week 28

Week 29

Week 30

# Week of July 5th to 10th

## Pmr and Amplifier

### Week Plan:

1. Plasmid construction of pET28a-AffiPmrB-aa.
2. Verification of AffiPmrB-A-C system by other methods like microplate reader.

### Progress:

1. Plasmid construction of pET28a-AffiPmrB-aa.
We failed to construct this plasmid.
2. Verification of AffiPmrB-A-C system by other methods like microplate reader.
We tried confocal again but didn't use microplate reader.

### Troubleshooting:

1. Plasmid construction of pET28a-AffiPmrB-aa.
Problem:
The PCR test of KanR-positive clone showed that they had a insertion fragment of about 1000bp or 2000bp instead of the correct length 3600bp.
Analysis:
We inferred that the enzyme restriction linearization didn't work well with our HiFi primer.
Solution:
We plan to change our HiFi primer and do the HiFi assembly again.
2. Verification of AffiPmrB-A-C system by other methods like microplate reader.
Problem:
The microplate reader which can test fluorescence was not available that week.

### Future Plan:

We planned to pause our experiment for a week and take the time to re-evaluate our project and rearrange further experiments.

## Nisin

### Week Plan:

1. Cotransformation of pNisAB and pNisC
2. Verify the expression of NisA-affi

### Progress:

1. Cotransformation of pNisAB and pNisC:
We successfully cotransform the two plasmids and the efficiency cotransformation was developed significantly.
2. Verify the expression of NisA-affi:
We used Ni column to purify NisA at first and did WB to verify the effect of purification. The concentration of NisA was low and the stripe of it in WB was inapparent.
![](https://static.igem.wiki/teams/4307/wiki/wikinotebookfiles/nisin/week2.jpg)

### Troubleshooting:

Failure of NisA purification:
Problem:
The concentration of NisA was low and the stripe of it in WB was inapparent.
Analysis:
Maybe the Ni column's ability of protein binding is insufficient.
Solution:
We planned to use new column to purify, we also planned to buy protein concentration column to concentrate the purified protein.

### Future Plan:

We planned to pause our experiment for a week and take the time to re-evaluate our project and rearrange further experiments.

## Logic Gate

### Week Plan:

1. Plasmid construction of v19. pUC-mCherry-attB-OR-attP-EGFP
2. Serine integrase function verification: preliminary experiment

### Progress:

1. Plasmid construction of v19. pUC-mCherry-attB-OR-attP-EGFP:
After weeks of efforts, we finally constructed this plasmid.
2. Serine integrase function verification
We co-transformed v18. pUC-attB-OR-attP-EGFP and v13. pET28a-ser integrase into BL21(DE3) strain to test for serine integrase function under IPTG induction.
We designed a pair of primers in the same direction before inversion, which would be in opposite directions after inversion, amplifying the sequences in between. Using this pair of primers, we tested whether serine integrase inverted the sequences between the two recombination sites.
We also sent the bacteria solution before and after inversion to sequencing for further confirmation.
![](https://static.igem.wiki/teams/4307/wiki/wikinotebookfiles/logicgate/3.png)

### Troubleshooting:

1. Leakage of serine integrase:
Problem:
The PCR result of sequences before and after inversion all turned out to be positive.
Analysis:
After communicating with Ba Fang, who had conducted related research on serine integrase and its recombination sites, we speculated that integrase had already been expressing right after transformation without the inducer due to leakage.
Solution:
Considering Ba Fang' s advice, we planned to examine the extent of leakage first in order to set a threshold for inversion verification. In other words, as long as stark contrast before and after the inversion can be established, the problem of leakage could be ignored.
2. Efficiency of inversion by serine integrase:
Problem:
The sequencing result of bacteria solution before and after inversion all showed that the sequences had NOT been inverted.
Analysis:
We speculated that sequences that had been inverted accounted for the vast majority, indicating that our induction was insufficient.
Solution:
We planned to optimize the induction condition in further experiments.
Future Plan:
We planned to pause our experiment for a week and take the time to re-evaluate our project and rearrange further experiments.

## HP

When constructing a two-component downstream logic system, we were inspired by the article published by Jian Li's team in 2022 (doi: 10.1093/nar/gkac124) and decided to use tryptophan integrase as the main control element for logic gating. Initially, we used a tryptophan integrase synthesized by the company, however, it was less efficient. In order to improve the efficiency of the tryptophan integrase, we contacted Professor. Jian Li to obtain the plasmid expressing the tryptophan integrase and its target of action mentioned in the article. Through Professor. Li, we contacted the first author, Mr. Fang Ba. He was kind enough to send us the plasmid and gave us advice on the design and details of our experiments.

## Education and Communication

This week, our team members visited Bluepha and had face-to-face communication to further understand the current state of the synthetic biology industry.

## Others

This week, we split into four groups and presented ideas for possible changes to our current project. The final vote was to adopt the toehold switch, a more responsive design that would hopefully solve the problems of our project; during the discussion, everyone was very involved and it seemed like a nostalgic return to the brainstorm state of the early days of the team.

# Week of July 11th to 17th

## Pmr and Amplifier

### Week Plan:

We planned to pause our experiment for a week and take the time to re-evaluate our project and rearrange further experiments.

### Progress:

We further consulted with Ba Fang, and proposed several solutions to tackle with the problems in our experiments.

### Troubleshooting:

1. Efficiency of inversion by serine integrase:
Problem:
The efficiency of inversion by serine integrase was too low to cause any difference before and after the induction.
Analysis:
We speculated that the low efficiency was due to improper induction condition or insufficient signal amplification.
Solution:
To be on the safe side, we changed part of our design and decided to use toehold switch-trigger RNA system as a possible substitute if the problem of serine integrase could not be solved.
2. Fluorescence intensity of AffiPmrB-A-C system:
Problem:
The fluorescence intensity of induced AffiPmrB-A-C system was too low, and was unstable showed by the fluorescence microscope.
Solution:
Verification again; try the variant AffiPmrB-aa; add amplifier to the system.

### Future Plan:

1. Plasmid construction of pBAD24-T7T3-LacZ-Toehold Switch 1 & pBAD24-T7T3-LacZ-Toehold Switch 2.
2. Plasmid construction of pET28a-mCherry-Toehold Switch 1 & pET28a-mCherry-Toehold Switch 2.
3. Construction of NisK/R-amplifier system.
4. Further verification of AffiPmrB-A-C system.

## Nisin

### Week Plan:

We planned to pause our experiment for a week and take the time to re-evaluate our project and rearrange further experiments.

### Progress:

We bought the protein concentration column. We decided to keep our original experiment plan after re-evaluation.
![](https://static.igem.wiki/teams/4307/wiki/wikinotebookfiles/nisin/week3.jpg)

### Future Plan:

1. Verify the expression of NisA-affi
2. Use microplate reader to detect the expression of EGFP of pNisinOE1

## Logic Gate

### Week Plan:

We planned to pause our experiment for a week and take the time to re-evaluate our project and rearrange further experiments.

### Progress:

We further consulted with Ba Fang, and proposed several solutions to tackle with the problems in our experiments.
![](https://static.igem.wiki/teams/4307/wiki/wikinotebookfiles/logicgate/4.png)

### Troubleshooting:

1. Leakage of serine integrase:
Problem:
Serine integrase started expressing even without induction due to leakage problem.
Analysis:
We speculated that the leakage may be reduced if we changed the expression and induction system.
Solution:
We planned to change to araC-pBAD system to express the serine integrase under arabinose induction given that araC-pBAD system had been proved of weak leakage in two-component system induction in previous work.
2. Efficiency of inversion by serine integrase:
Problem:
The efficiency of inversion by serine integrase was too low to cause any difference before and after the induction.
Analysis:
We speculated that the low efficiency was due to improper induction condition or insufficient signal amplification.
Solution:
We planned to try arabinose induction first. If failed, we planned to try to introduce a resource allocator or amplifier into our genetic circuit to further amplify the signal.
Future Plan:
1. Plasmid construction of v34. pBAD-ser integrase
2. Plasmid construction of v32. Final\_mCherry-OR-EGFP-pBAD-araC-cro-cI

## HP

This week we compiled our previous HP records, analyzed the suggestions made by our consultants, and began thinking about how to improve our program based on these suggestions. We have kept in touch with Mr. Fang Ba to continue to address the issues encountered on serine integrase.

## Education and Communication

This week we had the first workshop of TAP. In this workshop, the three teams gave an overview of their 2022 projects and discuss how the TAP teams can bridge the contexts of science and art to reflect and create together.

# Week of July 18th to 24th

## Pmr and Amplifier

### Week Plan:

1. Plasmid construction of pBAD24-T7T3-LacZ-Toehold Switch 1 & pBAD24-T7T3-LacZ-Toehold Switch 2.
2. Plasmid construction of pET28a-mCherry-Toehold Switch 1 & pET28a-mCherry-Toehold Switch 2.
3. Further verification of AffiPmrB-A-C system.

### Progress:

1. Plasmid construction of pBAD24-T7T3-LacZ-Toehold Switch 1 & pBAD24-T7T3-LacZ-Toehold Switch 2.
We prepared PCR fragments for HiFi assembly, and sent the LacZ-Toehold Switch 1&2 sequence to company for synthesis.
2. Plasmid construction of pET28a-mCherry-Toehold Switch 1 & pET28a-mCherry-Toehold Switch 2.
We sent the mCherry-Toehold Switch 1&2 sequence to company for synthesis.
3. Further verification of AffiPmrB-A-C system.
We tested the EGFP fluorescence intensity of AffiPmrB-A-C system by microplate reader.

### Troubleshooting:

1. Further verification of AffiPmrB-A-C system.
Problem:
The fluorescence intensity of EGFP didn't change much after being induced, and was unstable.
Analysis:
The augmenter of EGFP expressed was too low that the error in measurement would cause unstable results, meanwhile the system itself may not be that stable.
Solution:
We plan to add amplifier to the AffiPmrB-A-C system.

### Future Plan:

1. Plasmid construction of pBAD24-T7T3-LacZ-Toehold Switch 1 & pBAD24-T7T3-LacZ-Toehold Switch 2.
2. Plasmid construction of pET28a-mCherry-Toehold Switch 1 & pET28a-mCherry-Toehold Switch 2.
3. Plasmid construction of pET28a-AffiPmrB-aa.

## Nisin

### Week Plan:

1. Verify the expression of NisA-affi
2. Use microplate reader to detect the expression of EGFP of pNisinOE1

### Progress:

1. Verify the expression of NisA-affi
We did the purification twice and the stripe was a little bit more apparent than last time.
2. Use microplate reader to detect the expression of EGFP of pNisinOE1
The expression of EGFP was low.
![](https://static.igem.wiki/teams/4307/wiki/wikinotebookfiles/nisin/week4.jpg)

### Troubleshooting:

Problem:
The expression of EGFP was low.
Analysis:
IPTG inducement didn't work well.
Solution:
We planned to replace the T7 promoter into the constitutive promoter.

### Future Plan:

1. Replace the T7 promoter into the constitutive promoter
2. Use purified NisA to induce expression

## Logic Gate

### Week Plan:

1. Plasmid construction of v34. pBAD-ser integrase
2. Plasmid construction of v32. Final\_mCherry-OR-EGFP-pBAD-araC-cro-cI
3. Serine integrase function verification:qPCR

### Progress:

1. Plasmid construction of v34. pBAD-ser integrase:
We successfully constructed this plasmid.
2. Plasmid construction of v32. Final\_mCherry-OR-EGFP-pBAD-araC-cro-cI:
We successfully inserted mCherry fragment and the new promoter OR into pET28a-EGFP vector (v30). We also constructed the fragment of pBAD-araC-cro (v31).
3. Serine integrase function verification:
We examined the inversion rate of serin integrase by qPCR under IPTG induction.
![](https://static.igem.wiki/teams/4307/wiki/wikinotebookfiles/logicgate/5.png)

### Troubleshooting:

1. Inversion efficiency of serine integrase
Problem:
The ct value of the sample before and after induction differed by 3 ct, indicating that the concentration of inverted sequences was 8 times compared with the concentration of the original sequences.
Analysis:
Even though differences before and after induction did exist, but it was insufficient to establish any contrast on fluorescence.
Solution:
We planned to run qPCR again after constructing our new plasmid with pBAD promoter.
Future Plan:
1. Plasmid construction of v32. Final\_mCherry-OR-EGFP-pBAD-araC-cro-cI: insert pBAD-araC-cro and cI fragments
2. Serine integrase function verification: co-transform v34. pBAD-ser integrase and v30. pET28a-mCherry-OR-EGFP

## HP

We continued to improve serine integrase on the suggestions given by Mr. Fang Ba. In the meantime, we made preliminary design of the hardware and contacted relevant professors to ask for methods.

# Week of July 25th to 30th

## Pmr and Amplifier

### Week Plan:

1. Plasmid construction of pBAD24-T7T3-LacZ-Toehold Switch 1 & pBAD24-T7T3-LacZ-Toehold Switch 2.
2. Plasmid construction of pET28a-mCherry-Toehold Switch 1 & pET28a-mCherry-Toehold Switch 2.
3. Plasmid construction of pET28a-AffiPmrB-aa.
Progress:
1. Plasmid construction of pBAD24-T7T3-LacZ-Toehold Switch 1 & pBAD24-T7T3-LacZ-Toehold Switch 2.
We successfully constructed the two plasmids.
2. Plasmid construction of pET28a-mCherry-Toehold Switch 1 & pET28a-mCherry-Toehold Switch 2.
The sequence sent for synthesis hadn't finished yet.
3. Plasmid construction of pET28a-AffiPmrB-aa.
We failed in constructing this plasmid.

### Troubleshooting:

1. Plasmid construction of pET28a-AffiPmrB-aa.
Problem:
The PCR test of KanR-positive clone still showed that they had a insertion fragment of about 1000bp or 2000bp instead of the correct length 3600bp.
Analysis:
We inferred that there might be other unexpected homologous sequence on the vector.
Solution:
We decided to change our clone strategies.

### Future Plan:

1. Verification of LacZ-Toehold Switch 1&2.
2. Plasmid construction of pET28a-mCherry-Toehold Switch 1 & pET28a-mCherry-Toehold Switch 2.
3. Plasmid construction of pET28a-AffiPmrB-aa.

## Nisin

### Week Plan:

1. Replace the T7 promoter into the constitutive promoter and construct the pNisin1
2. Use purified NisA to induce expression
3. Express the NisA-affi

### Progress:

1. Replace the T7 promoter into the constitutive promoter:
We successfully replaced the promoter.
2. Express the NisA-affi:
We successfully expressed the NisA-affi and purified it.
3. Use purified NisA to induce expression
We used gradient concentration of nisin to induce expression of pNisinOE1, but the curve of expression can't be repeated.
![](https://static.igem.wiki/teams/4307/wiki/wikinotebookfiles/nisin/week5.jpg)

### Troubleshooting:

Problem:
Expression results can't be repeated
Analysis:
We used 12ml shaking tubes to do the expression, which made differences between groups, and the exact concentration of bacteria is different in each tube and each experiment.
Solution:
We planned to use conical flask to do the expression and detect the expression of EGFP by inducing it with a different time.

### Future Plan:

1. Expression of pNisin1
2. Constuct the pNisin2

## Logic Gate

### Week Plan:

1. Plasmid construction of v32. Final\_mCherry-OR-EGFP-pBAD-araC-cro-cI: insert pBAD-araC-cro and cI fragments
2. Serine integrase function verification: co-transform v34. pBAD-ser integrase and v30. pET28a-mCherry-OR-EGFP

### Progress:

1. Plasmid construction of v32. Final\_mCherry-OR-EGFP-pBAD-araC-cro-cI:
We inserted pBAD-araC-cro and cI fragments into the vector, and the final plasmid was sent to sequencing.
2. Serine integrase function verification:
The co-transformation of v34. pBAD-ser integrase and v30. pET28a-mCherry-OR-EGFP into BL21(DE3) strain failed.
![](https://static.igem.wiki/teams/4307/wiki/wikinotebookfiles/logicgate/6.png)

### Troubleshooting:

1. Failure of co-transformation
Problem:
Several rounds of co-transformation all failed. No colonies can be seen on the plate.
Analysis:
Such results indicated that plasmids failed to be included into the bacteria during co-transformation.
Solution:
We planned to optimize the co-transformation system by adjusting the amount of plasmids being transformed.
Future Plan:
1. Plasmid construction of v32. Final plasmid: confirm sequencing result
2. Serine integrase function verification: test inversion rate by qPCR under arabinose induction

## HP

We started with the overall design of the sperm test chip, but as the members were relatively new to hardware design and actual construction, we consulted Professor Chen, who has been an iGEM instructor for many years, for advice. During our consultation with Professor Guoqiang Chen, he mentioned that the cost of microfluidic chips might be an issue, and this is what we were reminded of during the hardware exchange with the BIT team: we should try to control the cost when designing microfluidic systems. In this regard, we approached Taiyan Zhou, who has extensive experience in microfluidic chip design, to ask him how to minimise costs.

## Education and Communication

This week we conducted the second workshop of TAP and invited four artists to share case studies of their artistic practices in the fields of bio-design, bio-art, and curation.

## Others

We started writing on our wiki this week.

## August

Week 31

Week 32

Week 33

Week 34

Week 35

# Week of August 1st to 7th

## Pmr and Amplifier

### Week Plan:

1. Verification of LacZ-Toehold Switch 1&2.
2. Plasmid construction of pET28a-mCherry-Toehold Switch 1 & pET28a-mCherry-Toehold Switch 2.

### Progress:

1. Verification of LacZ-Toehold Switch 1&2.
We failed in verifying LacZ-Toehold Switch 1&2.
2. Plasmid construction of pET28a-mCherry-Toehold Switch 1 & pET28a-mCherry-Toehold Switch 2.
We successfully constructed the plasmid pET28a-mCherry-Toehold Switch 2, while the sequence mCherry-Toehold Switch 1 still hadn't been synthesized.

### Troubleshooting:

1. Verification of LacZ-Toehold Switch 1&2.
Problem:
Nothing changed after X-gal and arabinose had been added.
Analysis:
We inferred that we hadn't successfully induced the promoter pBAD.
Solution:
We planed to test pBAD first and build a protocol of pBAD inducing.

### Future Plan:

1. Verification of LacZ-Toehold Switch 1&2.
2. Plasmid construction of pET28a-mCherry-Toehold Switch 1.
3. Verification of mCherry-Toehold Switch 2.

## Nisin

### Week Plan:

1. Expression of pNisin1
2. Constuct the pNisin2

### Progress:

1. Expression of pNisin1
We transformed pNisin1 into DH5α and induced it with nisin, but the plates were all white. No expression of lacZ was detected.
2. Constuct the pNisin2
The stripes in the gel after colony PCR were weird.
![](https://static.igem.wiki/teams/4307/wiki/wikinotebookfiles/nisin/week6.jpg)

### Troubleshooting:

1. Failure of expression:
Problem:
The plates were all white. No expression of lacZ was detected.
Analysis:
DH5α strain is not a good expression strain, the protein is not normally expressed or the expression amount is too little.
Solution:
We planned to replace lacZ into EGFP and use microreader to detect expression.
2. Colony PCR:
Problem:
The stripes in the gel after colony PCR were weird.
Analysis:
Maybe there is something wrong with the agarose gel we prepared.
Solution:
We send the plasmid to the company for sequencing.

### Future Plan:

1. Replace lacZ into EGFP and use microplate reader to detect expression.
2. Use NisA-affi to induce expression.

## Logic Gate

### Week Plan:

1. Plasmid construction of v32. Final plasmid: confirm sequencing result
2. Serine integrase function verification: test inversion rate by qPCR under arabinose induction

### Progress:

1. Plasmid construction of v32. Final plasmid:
Sequencing results confirmed that our plasmid construction was successful.
2. Serine integrase function verification:
We co-transformed v34. pBAD-ser integrase and v30. pET28a-mCherry-OR-EGFP into BL21(DE3) strain. Colony PCR results showed that our co-transformation was successful.
We inducted under different conditions (arabinose concentration, temperature, and time) to explore the optimal experiment condition for serine integrase to function.
We performed qPCR after arabinose induction to test the inversion rate of serine integrase, yet no significant difference before and after induction can be seen.
![](https://static.igem.wiki/teams/4307/wiki/wikinotebookfiles/logicgate/7.png)

### Troubleshooting:

1. qPCR results:
Problem:
No significant results can be seen before and after induction.
Analysis:
We speculated that the induction condition was still not optimal, thus insufficient amount of serine integrase can be expressed. In addition, the qPCR system was not rigorous as well.
Solution:
We planned to introduce an internal reference in qPCR.
Future Plan:
1. Serine integrase function verification: qPCR
2. Cro&cI function verification: fluorescence measurement by microplate reader

## HP

As for software, what we want to do is essentially a regression problem, and one of the datasets found now is the promoter sequences of prokaryotes and the expression levels of their corresponding genes. We hope to be able to predict the corresponding intensity given the promoter sequence by using some deep learning models. So we asked Zheni Zeng, a PhD student in the Natural Language Processing and Social Humanities Computing Laboratory (THUNLP) at Tsinghua University, for advice.

## Education and Communication

This week we had the third workshop of TAP. CAFA\_China presented their team's exhibition proposal and overall plan in detail, followed by a discussion and adjustments.

# Week of August 8th to 14th

## Pmr and Amplifier

### Week Plan:

1. Verification of LacZ-Toehold Switch 1&2.
2. Plasmid construction of pET28a-mCherry-Toehold Switch 1.
3. Verification of mCherry-Toehold Switch 1&2.

### Progress:

1. Verification of LacZ-Toehold Switch 1&2.
We successfully tested pBAD.
2. Plasmid construction of pET28a-mCherry-Toehold Switch 1.
We successfully constructed this plasmid.
3. Verification of pET28a-mCherry-Toehold Switch 1&2.
We failed in inducing the trigger RNA.

### Future Plan:

1. Verification of LacZ-Toehold Switch 1&2.
2. Verification of mCherry-Toehold Switch 1&2.
3. Construction of plasmid J23100-NisK-NisR-PnisA-T7T3-EGFP.

## Nisin

### Week Plan:

Because all our team members are not at school, this week's experiment is suspended.

### Progress:

![](https://static.igem.wiki/teams/4307/wiki/wikinotebookfiles/nisin/week7.jpg)

### Future Plan:

1. Replace lacZ into EGFP and use microplate reader to detect expression.
2. Use NisA-affi to induce expression.

## Logic Gate

### Week Plan:

1. Serine integrase function verification: qPCR
2. Cro&cI function verification: fluorescence measurement by microplate reader

### Progress:

1. Serine integrase function verification: qPCR
We co-transformed v34. pBAD-ser integrase and v30. pET28a-mCherry-OR-EGFP into BL21(DE3) strain. Colony PCR results showed that our co-transformation was successful.
We inducted under different conditions (arabinose concentration, temperature, and time) to explore the optimal experiment condition for serine integrase to function.
We performed qPCR after arabinose induction to test the inversion rate of serine integrase, yet no significant difference before and after induction can be seen.
2. Cro&cI function verification: fluorescence measurement by microplate reader
We transformed v32. Final plasmid into BL21(DE3) strain to test the function of cro and cI under arabinose induction.
We inducted under different conditions (arabinose concentration, temperature, and time) to explore the optimal experiment condition for cro and cI to function.
We measured fluorescence intensity by microplate reader after arabinose induction to test the function of cro and cI. cI seemed to function, yet cro had no significant result.
![](https://static.igem.wiki/teams/4307/wiki/wikinotebookfiles/logicgate/8.png)

### Troubleshooting:

1. qPCR result:
Problem:
No significant result can be seen before and after induction. We ran the qPCR product on the gel, but nothing showed on the gel.
Analysis:
We speculated that either one or both of the plasmids were lost after co-transformation. Antibiotic was supposed to be selective, but those bacteria without chloramphenicol resistance could still survive on some residual chloramphenicol in the solution. Another possibility was that pBad promoter or araC was not working.
Solution:
To tackle the issue of losing plasmids after co-transformation, we planned to construct the serine integrase and its recombination sites on the same plasmid, namely plasmid v41. Two in one. To test whether the pBad promoter could work, we planned to design a verification plasmid to test the function of pBad promoter.
2. Cro&cI function:
Problem:
Under the optimal induction condition, cI seemed to function well, but cro has no significant result.
Analysis:
Conclusions were too soon to be drawn before we repeated the experiment for several times.
Solution:
We planned to repeat this experiment.
Future Plan:
1. Cro&cI function verification: fluorescence measurement by microplate reader
2. Plasmid construction of Two in one plasmid
3. Plasmid construction of the promoter verification plasmid

## HP

Since we are making a product for the public, this week we published a questionnaire to ask potential users about their willingness to use it and the public acceptability of synthetic biology for home testing devices.

## Education and Communication

This week we had the fourth workshop of TAP, where our team presented our exhibition proposal and discussed with CAFA\_China and Peking team.

## Others

We attended the offline meeting held by iBowu and were very happy to finally meet iGEMers from other teams off-line!

# Week of August 15th to 21st

## Pmr and Amplifier

### Week Plan:

1. Verification of LacZ-Toehold Switch 1&2.
2. Verification of mCherry-Toehold Switch 1&2.
3. Construction of plasmid J23100-NisK-NisR-PnisA-T7T3-EGFP.

### Progress:

1. Verification of LacZ-Toehold Switch 1&2.
We failed in verifying LacZ-Toehold Switch 1&2.
2. Verification of mCherry-Toehold Switch 1&2.
We failed in verifying mCherry-Toehold Switch 1&2.
3. Construction of plasmid J23100-NisK-NisR-PnisA-T7T3-EGFP.
We prepared all PCR fragments for HiFi assembly.

### Future Plan:

1. Verification of LacZ-Toehold Switch 1&2.
2. Verification of pET28a-mCherry-Toehold Switch 1&2.
3. Construction of plasmid J23100-NisK-NisR-PnisA-T7T3-EGFP.

## Nisin

### Week Plan:

1. Replace lacZ into EGFP and use microplate reader to detect expression.
2. Use NisA-affi to induce expression.
3. Knock out lacZ of BL21(DE3)

### Progress:

1. Use NisA-affi to induce expression:
We used Nisin and NisA-affi to induce expression for a lot of times, but the results had big moves, we coundn't get a stable result.
2. Replace lacZ into EGFP and use microplate reader to detect expression:
We successfully constructed the plasmid.
3. Knock out lacZ of BL21(DE3)
We bought the CRISPR-Cas9 system and designed four guide RNAs for CRISPR-Cas9.
![](https://static.igem.wiki/teams/4307/wiki/wikinotebookfiles/nisin/week8.jpg)

### Troubleshooting:

Problem:
We used Nisin and NisA-affi to induce expression for a lot of times, but the results had big moves, we coundn't get a stable result.
Analysis:
Maybe the expression of EGFP was low which made microplate reader difficult to distinguish.
Solution:
We planned to use fluorescence microscope to observe EGFP expression directly

### Future Plan:

1. Use NisA-affi to induce expression.
2. Use fluorescence microscope to observe EGFP expression directly
3. Knock out lacZ of BL21(DE3)

## Logic Gate

### Week Plan:

1. Cro&cI function verification: fluorescence measurement by microplate reader
2. Plasmid construction of Two in one plasmid
3. Plasmid construction of the promoter verification plasmid

### Progress:

1. Cro&cI function verification: fluorescence measurement by microplate reader
We transformed v32. Final plasmid into BL21(DE3) strain to test the function of cro and cI under arabinose induction.
We inducted under different conditions (arabinose concentration, temperature, and time) to explore the optimal experiment condition for cro and cI to function.
We measured fluorescence intensity by microplate reader after arabinose induction to test the function of cro and cI. cI seemed to function well, yet cro had no significant result.
2. Plasmid construction of Two in one plasmid
We finished constructing v41. Two in one plasmid. Sequencing result confirmed that our construction was successful.
3. Plasmid construction of the promoter verification plasmid
We constructed v37. pBad-mCherry plasmid and v38. pBad-mCherryOR plasmid to test promoter function, but sequencing result failed.
![](https://static.igem.wiki/teams/4307/wiki/wikinotebookfiles/logicgate/9.png)

### Troubleshooting:

1. Cro&cI function:
Problem:
Under the optimal induction condition, cI seemed to function well, but cro has no significant result.
Analysis:
One possibility was that the pBad promoter was not working. Another possibility was that the cro fragment was not working.
Solution:
We planned to test the pBad promoter function first.
2. Failed sequencing result of the promoter verification plasmid:
Problem:
The sequencing of the bacteria solution turned out to be positive, but the sequencing of plasmid failed due to low concentration.
Analysis:
The concentration of DNA was indeed very low after plasmid extraction.
Solution:
We planned to repeat the construction from HiFi and transformation.
Future Plan:
1. Serine integrase function verification: qPCR and microplate reading
2. Plasmid construction of the promoter verification plasmid
3. Promoter function verification

## HP

We learned that Prof. Jinming Lin from Tsinghua University is dedicated to the research of microfluidics-based rapid microbial assays, including the development of instruments and kits related to the assay technology. In order to obtain more professional guidance on microfluidics, this week we contacted Prof. Jinming Lin via email. Professor Lin has a long history of teaching courses related to microfluidics and has extensive experience in guiding undergraduate students in their research.

## Education and Communication

This week we had the fourth workshop of TAP, this time it was the presentation of the exhibition of Peking team.

## Others

We were all very encouraged to learn that we had won three awards at CCiC!

# Week of August 22nd to 28th

## Pmr and Amplifier

### Week Plan:

1. Verification of LacZ-Toehold Switch 1&2.
2. Verification of mCherry-Toehold Switch 1&2.
3. Construction of plasmid J23100-NisK-NisR-PnisA-T7T3-EGFP.
4. Verification of NisK/R-amplifier.

### Progress:

1. Verification of LacZ-Toehold Switch 1&2.
We failed in verifying LacZ-Toehold Switch 1&2.
2. Verification of mCherry-Toehold Switch 1&2.
We successfully verified mCherry-Toehold Switch 1&2.
3. Construction of plasmid J23100-NisK-NisR-PnisA-T7T3-EGFP.
We successfully constructed this plasmid.
4. Verification of NisK/R-amplifier.
We successfully verified NisK/R-amplifier by EGFP fluorescence intensity using fluorescence microscope.

### Future Plan:

1. Verification of LacZ-Toehold Switch 1&2 (If it still remains no result, we would give up Toehold Switch).
2. Change the promoter of Toehold Switch-mCherry for a stronger one.
3. Verification of NisK/R-amplifier.

## Nisin

### Week Plan:

1. Use NisA-affi to induce expression.
2. Use fluorescence microscope to observe EGFP expression directly
3. Knock out lacZ of BL21(DE3)

### Progress:

1. Use NisA-affi to induce expression.
We used Nisin and NisA-affi to induce expression for a lot of times, but the results had big moves, we coundn't get a stable result.
2. Use fluorescence microscope to observe EGFP expression directly
We induced pNisin2 with 4 h and detected the fluorescence use flurescence microscope. The results were positive, but the induction effect was not ideal.
3. Knock out lacZ of BL21(DE3)
We failed to knock the gene, and we gave up due to the time limitation.
![](https://static.igem.wiki/teams/4307/wiki/wikinotebookfiles/nisin/week9.jpg)

### Troubleshooting:

1. The induction effect was not ideal
Problem:
We induced pNisin2 with 4 h and detected the fluorescence use flurescence microscope. The results were positive, but the induction effect was not ideal.
Analysis:
The PnisA promoter had relatively strong leakage, which made the induction effect inapparent.
Solution:
We planned to change the expression strain from BL21(DE3) to Mach1-T1, which was used widely for expression to improve the induction effect.
2. Failed to knock out the lacZ gene:
Problem:
We failed to knock the gene, and we gave up due to the time limitation.
3. Unstable expression curve
Problem:
We induced pNisin2 with 4 h and detected the fluorescence use flurescence microscope. The results were positive, but the induction effect was not ideal.
Analysis:
Maybe the NisK could not transfer to cell membrane well, which made it difficult to accept the signal of Nisin.
Solution:
We planned to construct a plasmid containing nisK which has EGFP downstream to observe the transfer of EGFP to cell membrane.

### Future Plan:

1. Use NisA-affi to induce expression.
2. Change the expression strain to Mach1-T1
3. Construct the plasmid containing NisK with EGFP downstream

## Logic Gate

### Week Plan:

1. Serine integrase function verification: qPCR and microplate reading
2. Plasmid construction of the promoter verification plasmid
3. Promoter function verification

### Progress:

1. Serine integrase function verification: qPCR and microplate reading
We transformed v41. Two in one plasmid into BL21(DE3) strain to test integrase function.
We inducted under various conditions (1% arabinose, different induction time, and different induction temperature) to explore the optimal induction condition for serine integrase to function.
We performed qPCR after arabinose induction to test the inversion rate of serine integrase, yet no significant difference before and after induction can be seen.
We also measured the fluorescence intensity using microplate reader to examine the inversion rate of serine integrase.
2. Plasmid construction of the promoter verification plasmid
We repeated the construction, and the sequencing result confirmed that our construction was successful. The two promoter verification plasmids (v37. & v38.) were ready for function verification.
3. Promoter function verification
We transformed v37. pBad-mCherry plasmid into BL21(DE3) strain. After 6h of arabinose induction, the bacteria turned red.
![](https://static.igem.wiki/teams/4307/wiki/wikinotebookfiles/logicgate/10.png)

### Troubleshooting:

1. Serine integrase function verification:
Problem:
The qPCR result showed that the difference between before and after induction was 1 ct at most. However, microplate reading showed significant contrast of fluorescence intensity before and after induction. We also discovered that the fluorescence intensity was already very high before induction at 16℃.
Analysis:
We speculated that our qPCR protocol might need to be improved. Apart from that, we speculated that there might be some leakage from pBad promoter or OR that resulted in the high background fluorescence level.
Solution:
We planned test whether there was any leakage from pBad promoter or OR using the promoter verification plasmid.
2. Cro&cI function:
Problem:
Under the optimal induction condition, cI seemed to function well, but cro has no significant result.
Analysis:
One possibility was that the pBad promoter was not working. Another possibility was that the cro fragment was not working.
Solution:
We planned to test the pBad promoter function first. If the promoter proved to be working, then we would replate the cro fragment.
Future Plan:
1. Promoter function verification
2. Plasmid construction of v40. Final with new cro plasmid
3. Serine integrase function verification: qPCR and microplate reading

## Education and Communication

This week we plan to conduct a road show in schools, using games and posters to educate the college community about the importance of reproductive health and how to maintain their own reproductive health.

## Others

This week, we finished the promotion video! It was done very close to the deadline, everyone worked hard.
![](https://static.igem.wiki/teams/4307/wiki/wikinotebookfiles/others/10.png)

Summer is almost over, and the students who went home are coming back to school, so we had another potluck this weekend!

# Week of August 29th to September 4th

## Pmr and Amplifier

### Week Plan:

1. Verification of LacZ-Toehold Switch 1&2 (If it still remains no result, we would give up Toehold Switch).
2. Change the promoter of Toehold Switch-mCherry for a stronger one.
3. Verification of NisK/R-amplifier.
4. Construction of plasmid pBAD-T7T3.
5. Verification of amplifier system: T7-T3.

### Progress:

1. Verification of LacZ-Toehold Switch 1&2 (If it still remains no result, we would give up Toehold Switch).
The induction remained no result: no color change after being induced and added X-gal.
2. Change the promoter of Toehold Switch-mCherry for a stronger one.
We found a proper promoter, but Toehold Switch was given up.
3. Verification of NisK/R-amplifier.
We verified NisK/R-amplifier by EGFP fluorescence intensity using microplate reader.
4. Construction of plasmid pBAD-T7T3.
We successfully constructed this plasmid.
5. Verification of amplifier system: T7-T3.
We verified T7-T3 system (induced by pBAD) by EGFP fluorescence intensity using microplate reader.

### Future Plan:

1. Verification of NisK/R-amplifier by FCM.
2. Construction of plasmid J23100-AffiPmrBAC-T7T3-EGFP.

## Nisin

### Week Plan:

1. Use NisA-affi to induce expression.
2. Change the expression strain to Mach1-T1
3. Construct the plasmid containing NisK with EGFP downstream

### Progress:

1. Use NisA-affi to induce expression.
The results looked better after changing the expression strain to Mach1-T1.
2. Change the expression strain to Mach1-T1
The Mach1-T1 strain had better performance in expressing EGFP and lacZ than BL21(DE3)
3. Construct the plasmid containing NisK with EGFP downstream(pcofocal)
We successfully construct the plasmid.
![](https://static.igem.wiki/teams/4307/wiki/wikinotebookfiles/nisin/week10.jpg)

### Future Plan:

1. Express pconfocol and detect the express of EGFP use fluorescence confocal microscope
2. Use NisA-affi to induce expression: continue
3. Change the PnisA promoter according to software results, and conduct expression.

## Logic Gate

### Week Plan:

1. Promoter function verification
2. Plasmid construction of v40. Final with new cro plasmid
3. Serine integrase function verification: qPCR and microplate reading

### Progress:

1. Promoter function verification
Results from microplate reading indicated that the pBad promoter was functioning well without any leakage. Our induction condition (1% arabinose, 37℃) proved to be effective. The results also indicated that OR probably did not have effect of promoting transcription.
2. Plasmid construction of v40. Final with new cro plasmid
We replaced the cro fragment in the final plasmid. The sequencing result confirmed that our construction was successful.
3. Serine integrase function verification: qPCR and microplate reading
We transformed v41. Two in one plasmid into BL21(DE3) strain.
We inducted under the optimal condition (1% arabinose, 37℃) to test serine integrase function.
We performed qPCR after arabinose induction to test the inversion rate of serine integrase, yet no significant difference before and after induction can be seen.
We also measured the fluorescence intensity using microplate reader to examine the inversion rate of serine integrase.
![](https://static.igem.wiki/teams/4307/wiki/wikinotebookfiles/logicgate/11.png)

### Troubleshooting:

1. qPCR result:
Problem:
No significant result can be seen for qPCR even though microplate reading indicated stark contrast of fluorescence intensity before and after the induction. Several wells even showed no ct after qPCR, yet correct bands can be seen after we ran the qPCR product on the gel.
Analysis:
We speculated that our qPCR system was somewhat inappropriate.
Solution:
We planned to optimize our qPCR protocol and system.
Future Plan:
1. Cro&cI function verification: test new cro
2. Serine integrase function verification: qPCR

## Education and Communication

We finished planning the roadshow this week, but unfortunately the school rejected our application for the event as it is now in the event insurance period.

## September

Week 36

Week 37

Week 38

Week 39

# Week of September 5th to 11th

## Pmr and Amplifier

### Week Plan:

1. Construction of plasmid J23100-AffiPmrBAC-T7T3-EGFP.
2. Verification of AffiPmrBAC system by FCM.

### Progress:

1. Construction of plasmid J23100-AffiPmrBAC-T7T3-EGFP.
We successfully constructed the plasmid J23100-AffiPmrBA.
2. Verification of AffiPmrBAC system by FCM.
We verified the AffiPmrBAC system by FCM and the augmenter of EGFP fluorescence intensity indeed existed but was very low.

### Future Plan:

1. Construction of plasmid J23100-AffiPmrBAC-T7T3-EGFP.
2. Verification of NisK/R-amplifier by FCM.

## Nisin

### Week Plan:

1. Express pconfocol and detect the express of EGFP use fluorescence confocal microscope
2. Use NisA-affi to induce expression: continue
3. Change the PnisA promoter according to software results, and conduct expression.

### Progress:

1. Express pconfocol and detect the express of EGFP use fluorescence confocal microscope
The result showed the transfer of NisK to cell membrane was normal, but there are large differences in different colonies.
2. Use NisA-affi to induce expression: continue
We had obtained a lot of data, some of which support our hypothesis.
3. Change the PnisA promoter according to software results, and conduct expression.
We successfully constructed the 10 plasmids(pRRH01-pRRH10)
![](https://static.igem.wiki/teams/4307/wiki/wikinotebookfiles/nisin/week11.jpg)

### Future Plan:

1. Induce pRRH01-pRRH10 and detect the expression of EGFP
2. Use flow cytometry to detect the expression of EGFP

## Logic Gate

### Week Plan:

1. Cro&cI function verification: test new cro
2. Serine integrase function verification: qPCR

### Progress:

1. Cro&cI function verification: test new cro
We transformed v40. Final with new cro plasmid into BL21(DE3) strain to test the function of cro and cI under arabinose induction.
We inducted under various conditions (1% arabinose concentration, different temperature, and time) to explore the optimal experiment condition for cro and cI to function.
We measured fluorescence intensity by microplate reader after arabinose induction to test the function of cro and cI.
2. Serine integrase function verification: qPCR
We repeated the experiment for several times only to receive similar results. Therefore, we consulted with other advisors and planned to optimize our qPCR protocol and system.
![](https://static.igem.wiki/teams/4307/wiki/wikinotebookfiles/logicgate/12.png)

### Troubleshooting:

1. Cro&cI function verification:
Problem:
Under the optimal induction condition, cI and cro both seemed to function. However, cro seem to be less effective than the positive control.
Analysis:
Conclusions were too soon to be drawn before we repeated the experiment for several times.
Solution:
We planned to repeat this experiment.
2. qPCR result:
Problem:
No significant result can be seen for qPCR even though microplate reading indicated stark contrast of fluorescence intensity before and after the induction. Several wells even showed no ct after qPCR, yet correct bands can be seen after we ran the qPCR product on the gel.
Analysis:
We speculated that our qPCR system was somewhat inappropriate.
Solution:
We planned to optimize our qPCR protocol and system.
Future Plan:
1. Cro&cI function verification: microplate reading to test new cro
2. Serine integrase function verification: qPCR

# Week of September 12th to 18th

## Pmr and Amplifier

### Week Plan:

1. Construction of plasmid J23100-AffiPmrBAC-T7T3-EGFP.
2. Verification of NisK/R-amplifier by FCM.
3. Construction of plasmid pBAD24-EriaCFP.

### Progress:

1. Construction of plasmid J23100-AffiPmrBAC-T7T3-EGFP.
We successfully constructed this plasmid (tested by PCR), and the plasmid was sent for sequencing.
2. Verification of NisK/R-amplifier by FCM.
We verified the NisK/R-amplifier, the result showed that the NisK/R system didn't work well with the T7T3 amplifier system.
3. Construction of plasmid pBAD24-EriaCFP.
We successfully constructed this plasmid and expressed EriaCFP.

### Future Plan:

1. Verification of AffiPmrBAC-amplifier system.
2. Build a colourimetric card of EriaCFP and mCherry.

## Nisin

### Week Plan:

1. Induce pRRH01-pRRH10 and detect the expression of EGFP
2. Use flow cytometry to detect the expression of EGFP

### Progress:

1. Induce pRRH01-pRRH10 and detect the expression of EGFP
The results showed the new promoters were stronger than the original promoter, and the effect of induction improved at the same time.
2. Use flow cytometry to detect the expression of EGFP
The results are positive and the data is roughly consistent with our assumptions.
![](https://static.igem.wiki/teams/4307/wiki/wikinotebookfiles/nisin/week12.jpg)

# Week of September 19th to 25th

## Pmr and Amplifier

### Week Plan:

1. Verification of AffiPmrBAC-amplifier system.
2. Construction of plasmid pET28a-AffiPmrB-aa.
1. Verification of AffiPmrBAC-amplifier system.
We verified the AffiPmrBAC-amplifier by EGFP fluorescence intensity using microplate reader, it showed a much better result than AffiPmrBAC after 10h induction.
2. Construction of plasmid pET28a-AffiPmrB-aa.

### Future Plan:

1. Verification of AffiPmrB-aaAC.

## Nisin

### Week Plan:

We have entered into a collaboration with HUST-China, and we will help them with the design of the experimental system. For this purpose, we need to help them to conduct induction experiments and observe the fluorescence by fluorescence microscopy.

### Progress:

This week, we completed an induction experiment with the bacteria provided to us by HUST-China. The bacteria were shaken to saturation overnight and inoculated into LB medium at a ratio of 1:50, and were induced by adding 0.5 mM IPTG and 50 μM Cu2+ at an OD of 0.6. After overnight induction, we centrifuged the organisms and washed them with PBS, and added primary and secondary antibodies for immunofluorescence reaction, and finally added 100 μL of PBS for filming and observation under fluorescence microscope.

### Troubleshooting:

In the results of this induction experiment, the number of fluorescent bacteria under the microscope was not very high, probably because the FITC-labeled secondary antibodies were not incubated strictly against light during the experiment. We will improve this in the next experiment.

### Future Plan:

We repeated the experiment and gave the results to HUST-China. we also discussed with them this week the results of the nisin induction experiment they performed for us.

# Week of September 26th to October 2nd

## Pmr and Amplifier

### Week Plan:

1. Verification of AffiPmrB-aaAC.

### Progress:

1. Verification of AffiPmrB-aaAC.
Weerified the AffiPmrB-aaAC by EGFP fluorescence intensity using microplate reader, it showed a better result than AffiPmrBAC: a higher augmenter of EGFP fluorescence intensity and a better affinity with IgG.

### Future Plan:

Wetlab finished.

## October

Week 40

Week 41
>

# Week of October 3rd to 9th

We worked through this week to complete our wiki, it feels good to be chased by the wiki freezes deadline!

# Week of October 10th to 12th

Here we comes to the end, iGEM brought us far more than we originally thought. We love this trip!

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
{% block page\_content %}

# Engineering

* Engineering
* [Overview](#P1)
* [Engineering of Two-Compoment System](#P2)
* [Engineering of Logic Gate](#P3)
* [Engineering of Software](#P4)
* [Engineering of Hardware](#P5)
* [References](#P6)

# Overview

In the process of our project development, we performed many engineering cycles to improve our design and experimental implementation. Here we present our engineering progress in three sections: engineering of two-component system, engineering of logic gate, and engineering of software. For each engineering process, we record it following the four general stages (design, build, test, and learn) to demonstrate our improvement.

![engineering-image-front.jpg](https://static.igem.wiki/teams/4307/wiki/wikiengineeringfiles/engineering-image-front.jpg)

![Schematic diagram of our engineering cycles. ](https://static.igem.wiki/teams/4307/wiki/wikiengineeringfiles/engineering-cycles.jpg)

**Fig.1 Schematic diagram of our engineering cycles.**

# Engineering of Two-Compoment System

### Signal amplification

#### Cycle 1: Weak fluorescence signal

##### Design

At first, we used *EGFP* as the reporter gene. It was placed under the promoter of the two-component system, so that if the two-component system worked, we could see the green fluorescence.

##### Build

The cloning of *EGFP* under the promoter of the Nisin two-component system was successful. Then it was transformed into the *E. coli* BL21 strain. Nisin was added to induce the expression of *EGFP*.

##### Test

Unfortunately, results showed that the signal was too weak to be seen even under the fluorescence microscopy.

##### Learn

We speculated that the promoter of the two-component system was so weak that it was almost impossible to observe the expression of downstream *EGFP* by naked eyes. Nevertheless, our goal was that the signal produced by the engineered *E. coli* could be readily observed at home. Therefore, we designed three ways to amplify our signal.

#### Cycle 2: Three ways of signal amplification

##### Design

The first way of signal amplification was to use amplifiers. We intended to empower the polymerase system without laying too much metabolic pressure on our engineered *E. coli*. Therefore, we chose to introduce the σ of T3 polymerase and the core of the T7 polymerase. This combination ensured higher level of transcription while limiting the toxic effect to a manageable extent.

The second way of signal amplification was to enhance the promoter strength of the two-component system. We designed a software to predict the strength of mutant promoters. Related details are described on [Software page](https://2022.igem.wiki/tsinghua/software).

The third way of signal amplification was to use a sensitive and readily-discerned reporter gene. We chose *lacZ* in place of *EGFP*, considering that a small amount of *lacZ* expression can lead to evident change of color.

##### Build

To implement the first plan, we successfully introduced the σ of T3 polymerase and the core of the T7 polymerase into the *E. coli* BL21 strain.

To implement the second plan, we successfully designed a software to predict the strength of mutant promoters.

To implement the third plan, we successfully replaced *EGFP* with *lacZ* as the reporter gene.

##### Test

To test our first solution, we demonstrated the efficiency of the amplifier by fluorescence spectrophotometry.

To test our second solution, we selected the top10 strongest promoters according to our software prediction and tested their promoter intensity respectively. We demonstrated that our software possessed a 90% accuracy in predicting promoter strength.

To test our third solution, the induction result of the two-component system after replacing *EGFP* with *lacZ* could be easily detected by observing the blue color.

##### Learn

Firstly, we demonstrated that the amplifier could fit in our gene circuit to amplify signals efficiently.

Secondly, we learned that we could use the mutant promoters with more enhanced strength to improve our product.

Thirdly, we found out that LacZ is suitable for signal representation for household devices. It can magnify faint signals, and the depth of color can also represent the strength of signals.

#### Cycle3: *lacZ* knock out

##### Design

Since we intended to select *lacZ* as a reporter gene downstream of the two-component system, the presence of *lacZ* in the genome of the strain BL21(DE3) would interfere with our design. Therefore, we attempted to knock out the *lacZ* gene in the BL21(DE3).

##### Build

We applied the CRISPR-Cas system to perform genomic knockouts. Specifically, we chose the CRISPR/AID system, a tool capable of converting cytosines to thymine (C-T) at 18-21 bp upstream of the NG/NGG-PAM sequence, to complete the mutation. We found six CAA/ CAG/ CGA codons in the *lacZ* gene of the BL21(DE3) genome, and four of these codon classes have NGG sequences at 18-21 bp upstream, which can be used as sites for designing gRNAs. We cloned these four gRNAs into the scaffold of gRNA in the gRNA plasmid by Hifi. Sequencing results verified that the cloning of gRNA-2 and gRNA-4 were successfully constructed.

##### Test

We co-transformed the successfully constructed gRNA plasmid with dCas9 plasmid into BL21(DE3) and coated the plate on petri dish containing X-gal for screening. The BL21(DE3) that was not transformed was also coated as a positive control.

##### Learn

After one day of incubation, the colonies growing on the petri dish were all blue, similar to the plates of the positive control, indicating that our LacZ knockdown failed. The reason may be that the gRNA we used was not efficient and caused off-target. We should try more gRNAs for knockdown at the same time.

### Response Time Reduction

#### Cycle1: Constitute Promoter

##### Design

Initially, *nisK* and *nisR* were placed under the T7 promoter and induced by IPTG. However, it took time for *nisK* and *nisR* to be expressed and embedded in the membrane, only after which the induction of the two-component system was possible. Consequently, there would be a lag between the adding of Nisin and the representation of signals. Therefore, we decided to replace the T7 promoter with a constitute promoter, rendering advanced expression of *nisK* and *nisR*.

##### Build

We successfully replaced the T7 promoter with a constitute promoter.

##### Test

The constitute promoter allowed for advanced expression of *nisK* and *nisR*, so that the induction of the two-component system could take place immediately after adding Nisin.

##### Learn

Replacing inducible promoter with constitute promoter can shorten the response time of Nisin induction, thus reducing the response time of the whole detection process of sperm quality examination.

#### Cycle2: Simultaneous induction

##### Design

Initially, we added IPTG first to induce the expression of *nisK* and *nisR*, then Nisin to induce the two-component system pathway. However, it took a long time to separate the induction into two stages. Therefore, we intended to add IPTG and Nisin together so that the experiment period could be shortened.

##### Build

We added IPTG and Nisin together to induce the expression of *nisK/nisR* and the two-component system pathway simultaneously.

##### Test

The result of simultaneous induction was basically the same with separate induction, while the former took shorter time.

##### Learn

The induction of the expression of on-membrane receptors of the two-component system and the induction of the two-component system pathway can be conducted simultaneously, reducing the experiment time without influencing the effect.

### Affi-PmrB with more amino acids retained

##### Design

Our previous modification of PmrB was to accurately replace its extracellular domain with the double-α-helix affibody recognizing Fc fragment. However, though Affi-PmrB/A can activate the expression of downstream EGFP under IgG induction, the result showed by microplate reader did not reveal an expected kinetic feature of Affi-PmrB’s function as a receptor. The result of molecular docking showed that the domain of Affi-PmrB interacting with Fc fragment overlapped with its transmembrane domain by 1-2 turns of α-helix, so we inferred that this might be the reason why Fc did not have a proper interaction with our Affi-PmrB.

##### Build

To solve this problem, we designed a new Affi-PmrB variant with more amino acids from PmrB retained. The amino acid 34-37 and 62-64 of PmrB, which exactly flank the transmembrane domain at the outer-membrane compartment, were retained to separate the recognition domain and the transmembrane domain.

##### Test

The prediction made by AlphaFold2 showed that the new variant also had a structure similar to PmrB. Meanwhile, the recognition and the transmembrane domain separated well according to result of molecular docking.The new Affi-PmrB variant with more amino acids was constructed into Affi-PmrB/A system and induced with IgG. Then we applied fluorescence spectrophotometry to measure the fluorescence intensity by microplate reader. Our results confirmed that the fluorescence intensity was much higher in the experimental group with antibody induction.This affi-pmrB variant seems improve the affinity between receptor and antibody.

![ Fluorescence spectrophotometry of Affi-PmrB/A system.  ](https://static.igem.wiki/teams/4307/wiki/wiki-result-files/pmr/fig8.png)

**Fig 2. Fluorescence spectrophotometry of Affi-PmrB/A system.**
Plasmid **pAffi-PmrB/A** was induced with IPTG (final concentration 0.24 mg/mL). The experimental group was then induced with 100 mM human IgG antibody, while the control group did not add any antibody. Fluorescence intensity of EGFP was measured by microplate reader. Averaged results from parallel repetition groups were recorded.

##### Learn

Through this cycle, we have a deeper understanding of the relationship between the structure and function of affi-pmrB receptor.

# Engineering of Logic Gate

### Two in one plasmid construction

#### Cycle 1

##### Design

At first, we planned to construct two plasmids, one containing serine integrase and the other containing verification sequences, using pET28a as the backbone with different antibiotic resistance.

##### Build

By reducing the concentration of two antibiotics to half of its working concentration, we obtained enough bacteria containing the two plasmids from co-transformation.

##### Test

We induced serine integrase at different temperatures for different time. Sequencing results indicated that the sequence between attP and attB had not been inverted in both induced and uninduced bacteria solution. In contrast, colony PCR using a pair of primers which can only anneal to inverted sequence suggested that all the bacteria contained inverted sequence. We speculated that the reasons behind such inconsistency might be:

* Leakage from the lacZ operator resulted in inverted sequence in uninduced bacteria.
* The expression level of serine integrase might be too low for inverted sequence to be detected by Sanger sequencing.

To find out the differences between the induced and uninduced bacteria, we conducted qPCR using the primers which can only anneal to inverted sequence. We found that for induced bacteria, less Ct was needed (about 3 Ct) for reaching the threshold, indicating that our induction was successful.

##### Learn

We learned that a promoter with less leakage would be more suitable for our system. Moreover, we should use a more sensitive method (for example, qPCR) to detect the effect of serine integrase.

#### Cycle 2

##### Design

We changed the backbone for serine integrase to pBad24 to reduce leakage. Then we measured the inversion rate of integrase by qPCR and fluorescence spectrophotometry after co-transformation.

##### Build

We cloned two kinds of serine integrase in pBad24, one was the same as the one in cycle 1 and [the other was a gift from Fang Ba](https://2022.igem.wiki/tsinghua/human-practices). We conducted experiments using the orignal intergase first.

##### Test

Uncertain about whether the tiny difference in fluoresence intensity could be detected, we conducted qPCR first. However, no stable results that matched our expectation could be obtained. Gel electrophoresis of qPCR products showed nothing, and SDS-PAGE of bacteria solution indicated no difference between induced and uninduced ones. After consulting with our advisor Xiaofei Ge, we proposed three possible reasons to explain our failure:

* The PBAD promoter on the backbone may be malfunctioning.
* The induction condition may not be the optimal one.
* Co-transformation might not work because pBad24 and pET28a have the same origin of replication.

We planned to verify the PBAD promoter, explore the optimal induction condition, and try to place all the gene elements on one single plasmid to avoid co-transformation in the next cycle.

##### Learn

We learned that plasmids with the same origin of replication could hardly exist in the same bacterial cell. We also learned more experience of troubleshooting.

#### Cycle 3

##### Design

We combined the two plasmids into one single plasmid and measured the effect of integrase using qPCR and fluorescence spectrophotometry. To verify the PBAD promoter intensity, we placed mCherry in the MCS of an empty pBad24 to characterize the promoter intensity. We also constructed another plasmid containing mCherry-OR sequence to see if OR sequence would influence the expression of integrase. We intended to measure mCherry fluorescence of the bacteria containing one of these two plasmids with or without arabinose induction.

##### Build

We successfully constructed the plasmid containing both integrase and verification sequence of intergase (pLogic 1), and the two plasmids for promoter verification.

##### Test

Fluorescence spectrophotometry results indicated that the PBAD promoter worked well with minor leakage, and OR sequence had little influence on the expression of downstream reporter gene. Characterization of integrase using pLogic1 was also successful by fluorescence spectrophotometry, but not qPCR.

##### Learn

We learned that combining two plasmids into one can be an efficient way to avoid co-transformation. Moreover, we were still striving to improve our protocols for qPCR.

### Replacement of *cro*

##### Design

In our previous design, we used the *cro* sequence from HUST 2016 iGEM project ([BBa\_K2036027](http://parts.igem.org/Part%3ABBa_K2036027)), which has been validated to work with the cI repressor to interoperate. However, in the course of our experiments, the derepression effect of Cro was always unsatisfactory. Consequently, we considered replacing the *cro* sequence with another homologous one.

##### Build

In a paper on bacterial memory elements[1], we found another homologous but slightly different *cro* sequence. We synthesized the sequence and replaced the original *cro* sequence with the new one in the plasmid by HiFi cloning.

##### Test

We used the same protocol as the previous experiments to verify the functionality of Cro again.

##### Learn

The results demonstrated that the new Cro was more suitable for our system. So we chose this version of Cro in the final design of pLogic2.

# Engineering of Software

#### Cycle 1: Random Forest → RNN

##### Design

We first attempted to apply the traditional machine learning model to predict the promoter strength of a given sequence. To be more specific, we applied the random forest model.

##### Build

We built the random forest model for our data set based on the skicit-learn library.

##### Test

However, no sound result was obtained because we could not determine which features were important when manually selecting features using the traditional machine learning model.

##### Learn

We realized that the traditional learning model was not feasible. Therefore, we hoped to try the deep learning method.

#### Cycle 2: RNN → LSTM

##### Design

Since our goal was to extract information from the promoter sequence, we considered employing the RNN model with sequential characteristics, which may improve the original effect.

##### Build

We built the RNN model for our data set based on Pytorch.

##### Test

After testing, the results outperformed the original one, but still failed to meet our expectation.

##### Learn

We speculated that this was likely due to the gradual decrease in the weight of bases far from the transcription start point during the parameter update process.

#### Cycle 3: LSTM → Transformer

##### Design

In order to obtain better results, we improved the RNN model to the LSTM model. The LSTM model could capture the information of the base in the promoter sequence far from the starting point, which is more conducive to information extraction.

##### Build

We built the LSTM model for our data set based on Pytorch.

##### Test

After testing, the results outperformed the RNN model, but we desired for better results.

##### Learn

Essentially, the LSTM model was still a variant of the RNN model. However, due to the sequential nature of its parameter update process, its parallelism was poor.

#### Cycle 4: Transformer → Transformer\_Encoder

##### Design

We searched for methods with higher parallelism to break through the resctrictions. Therefore, we turned our attention to Transformer, a model with higher amount of parameters and higher representation power.

##### Build

We built the Transformer model for our data set based on Pytorch.

##### Test

After testing, the results outperformed the LSTM model, but the running speed was much slower.

##### Learn

The Transformer model was originally suitable for machine translation tasks, but in practice it was not fully applicable to our biological problem. The Decoder structure in Transformer may not be necessary in our problem, which may affect the training speed.

#### Cycle 5: Transformer\_Encoder → Transformer\_Encoder+Dropout

##### Design

In order to make the Transformer model more suitable for this biological problem, we chose to use only the Encoder structure in the Transformer, which was good enough to extract features.

##### Build

We built the model of Transformer\_Encoder for our data set based on Pytorch.

##### Test

After testing, the results outperformed the precious model, but the phenomenon of overfitting always occurred.

##### Learn

We introduced the Dropout mechanism to solve the problem of overfitting and received wonderful results.

**For more detailed explanation of the abovementioned model, please visit our  [Modeling page.](https://2022.igem.wiki/tsinghua/model)**

# Engineering of Hardware

### Cycle: Pressure pump drive → Finger-pressing pump drive

#### Design

At first, we used traditional microfluidic technology, which drove the liquid flow by external pressure pump. The gradient generator composed of serpentine channels was located on both sides of the main channel. And this design made the chemokine solution flow in from one side and flow out from the other side.

![The first version of chip design drawing(A) and chip(B) ](https://static.igem.wiki/teams/4307/wiki/wikiengineeringfiles/engineering-hard-1.png)

**Fig.2 The first version of chip design drawing(A) and chip(B)**

But this microfluidic chip need external devices to provide power for the flow of liquid, which would bother our customers if they need to connect external devices for the microfluidic chip at home. In order to solve the problem, liquids with different concentrations of chemokine will be driven by a finger-pressing pump to form a concentration gradient. The chemokine loading wells in the gradient generator became much larger to fit in the size of human fingers, and only one side of gradient generator was retained to make the chip small enough for household use.

#### Build

After determining the structure of the mcrofluidic chip, the channel was built using PolyJet 3D printing technology via a Stratasys Connex3 Objet260 3D printer.

![The final version of chip design drawing(A) and chip(B) ](https://static.igem.wiki/teams/4307/wiki/wikiengineeringfiles/engineering-hard-2fifinal.png)

**Fig.3 The final version of chip design drawing(A) and chip(B)**

#### Test

The chip of new design can still form concentration gradient, and the liquid flow out of two finger-pressing pumps can fill in the overall channels of our chip.

#### Learn

We introduced the finger-pressing Pump to make our products more convenient for customers.

# References

[1] Kotula, J. W., Kerns, S. J., Shaket, L. A., Siraj, L., Collins, J. J., Way, J. C., & Silver, P. A. (2014). Programmable bacteria detect and record an environmental signal in the mammalian gut. *Proceedings of the National Academy of Sciences of the United States of America, 111*(13), 4838–4843. https://doi.org/10.1073/pnas.1321321111

© 2022 - Content on this site is licensed under a [Creative Commons Attribution 4.0 International license](https://creativecommons.org/licenses/by/4.0/).

The repository used to create this website is available at [gitlab.igem.org/2022/tsinghua](https://gitlab.igem.org/2022/tsinghua).

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

## Inspirations

---

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

# Results

* Results
* [Affi-PmrB/A System](#P2)
* [Affi-NisK/R System](#P3)
* [Logic Gate](#P4)
* [Reporter System](#P5)

**Here we present our wetlab results in four sections: TCS1 Affi-PmrB/A system, TCS2 Affi-NisK/R system, logic gate, and reporter system.**

# Affi-PmrB/A System

### Structure Prediction

In order to determine the feasibility of our modification, we first used AlphaFold2 software to predict the structure of the receptor Affi-PmrB that we constructed. Compared with the original receptor PmrB (uniport ID: P36557 · BASS\_SALTY), the predicted structure of Affi-PmrB according to our modified sequences are highly similar. Almost identical structure is displayed in the recognition area of the receptor and ligand, confirming the feasibility of our modification.

![(A)The predicted structure of Affi-PmrB by AlphaFold2. (B) The structure of PmrB. ](https://static.igem.wiki/teams/4307/wiki/wiki-result-files/pmr/fig1.png)

**Fig 1. (A)The predicted structure of Affi-PmrB by AlphaFold2. (B) The structure of PmrB.**

Molecular docking was simulated between the modified receptor Affi-PmrB and the Fc peptide of IgG antibody to further determine the feasibility of our modification scheme. We downloaded the Fc peptide structure (PDB DOI: 10.2210/pdb5VGP/pdb) from the PDB database, and then used the online protein molecular docking software Vasklab. Simulation results demonstrated that the Fc peptide could still bind to the ligand recognition region of our modified receptor with hydrophobic bonds, indicating that our modification did not affect the binding of Fc peptide with affibody.

![Molecular docking simulation results. ](https://static.igem.wiki/teams/4307/wiki/wiki-result-files/pmr/fig2.png)

**Fig 2. Molecular docking simulation results. (A) The predicted structure of Affi-PmrB. (B) The Fc region of IgG. (C) The simulation of molecular docking between Fc region and Affi-PmrB predicted by Vasklab.**

In summary, the strutural prediction and molecular docking simulation results have verified the feasibility of our modification design, confirming that the modified receptor Affi-Pmr still possesses the correct structure capable of binding and interactering with the Fc region of IgG antibody. Therefore, we have demonstrated that theoretically our modified receptor Affi-Pmr can serve as a protein signal detector.

### Plasmid Construction

This section presents the success of our plasmid construction. The cloning strategies and construction results for each plasmid are shown as follows. Detailed protocols can be found on the [Method page](https://2022.igem.wiki/tsinghua/methods).

#### pAffi-PmrB/A

##### Plasmid Design

**pAffi-PmrB/A** has the backbone of pET28a, carrying:

* designed sequence to express Affi-PmrB
* gene sequence to express PmrA
* IPTG-inducible promoter, driving the expression of Affi-PmrB and PmrA
* promoter PmrC, driving the expression of downstream reporter gene
* EGFP as the reporter gene
* resistance marker KanR for selection

![ Plasmid design of pAffi-PmrB/A. ](https://static.igem.wiki/teams/4307/wiki/wiki-result-files/pmr/fig3.png)

**Fig 3. Plasmid design of pAffi-PmrB/A.**

##### Cloning Strategies

PCR with high fidelity Q5 polymerase was conducted to amplify fragments and linearize vectors. Fragments and linearized vectors were assembled by NEBuilder® HiFi DNA assesmbly.

##### Construction Results

* Affi-PmrB-PmrA
* PmrC
* EGFP

![ Construction result of pAffi-PmrB/A. ](https://static.igem.wiki/teams/4307/wiki/wiki-result-files/pmr/fig4.png)

**Fig 4. Construction result of pAffi-PmrB/A.**

#### pAffi-PmrB/A-amplifier

##### Plasmid Design

**pAffi-PmrB/A-amplifier** has the backbone of pET28a, carrying:

* designed sequence to express Affi-PmrB
* gene sequence to express PmrA
* IPTG-inducible promoter, driving the expression of Affi-PmrB and PmrA
* promoter PmrC, driving the expression of T3 σ
* T7 core
* EGFP as the reporter gene
* resistance marker CmR for selection

![ Plasmid design of pAffi-PmrB/A-amplifier. ](https://static.igem.wiki/teams/4307/wiki/wiki-result-files/pmr/fig5.png)

**Fig 5. Plasmid design of pAffi-PmrB/A-amplifier.**

##### Cloning Strategies

PCR with high fidelity Q5 polymerase was conducted to amplify fragments and linearize vectors. Fragments and linearized vectors were assembled by NEBuilder® HiFi DNA assesmbly. Amplifier was added to plasmid pAffi-PmrB/A.

##### Construction Results

* Affi-PmrB-PmrA
* PmrC
* T7T3 amplifier-EGFP

![ Construction result of pAffi-PmrB/A-amplifier. ](https://static.igem.wiki/teams/4307/wiki/wiki-result-files/pmr/fig6.png)

**Fig 6. Construction result of pAffi-PmrB/A-amplifier.**

### Wetlab Verification

#### Characterization of Affi-PmrB/A system

We transformed plasmid **pAffi-PmrB/A** into *E. coli* for verification. IPTG was used to induce the expression of Affi-PmrB receptor and PmrA protein, and human IgG antibody was added to the experimental group. The fluorescence of EGFP was observed under fluorescence microscope. Our results demonstrated that the fluorescence signal was much stronger with antibody induction, indicating that our Affi-PmrB/A system can efficiently detect and respond to target protein signals.

![ Fluorescence microscopy of Affi-PmrB/A system. ](https://static.igem.wiki/teams/4307/wiki/wiki-result-files/pmr/fig7.png)

**Fig 7. Fluorescence microscopy of Affi-PmrB/A system.**
Plasmid **pAffi-PmrB/A** was induced with IPTG (final concentration 0.24 mg/mL). The experimental group (B and D) was then induced with 100 mM human IgG antibody, while the control group (A and C) did not add any antibody. Photos were taken under confocal fluorescence microscope (60×).

To increase fluorescence intensity, we designed a new Affi-PmrB variant with more amino acids from PmrB retained. The amino acid 34-37 and 62-64 of PmrB, which exactly flank the transmembrane domain at the outer-membrane compartment, were retained to separate the recognition domain and the transmembrane domain. Then we applied fluorescence spectrophotometry to measure the fluorescence intensity by microplate reader. Our results confirmed that the fluorescence intensity was much higher in the experimental group with antibody induction.

![ Fluorescence spectrophotometry of Affi-PmrB/A system.  ](https://static.igem.wiki/teams/4307/wiki/wiki-result-files/pmr/fig8.png)

**Fig 8. Fluorescence spectrophotometry of Affi-PmrB/A system.**
Plasmid **pAffi-PmrB/A** was induced with IPTG (final concentration 0.24 mg/mL). The experimental group was then induced with 100 mM human IgG antibody, while the control group did not add any antibody. Fluorescence intensity of EGFP was measured by microplate reader. Averaged results from parallel repetition groups were recorded.

#### Characterization of Affi-PmrB/A-amplifier system

Although the function of Affi-PmrB/A system had been verified, the fluorescence signal output by the engineered bacteria was very weak. Since our goal was to observe the fluorescence signal with naked eyes, we introduced the T7-T3 cascade amplifier into our Affi-Pmr system to amplify the output fluorescence signal output. The fluorescence spectrophotometry results demonstrated the effectiveness of signal amplification since the increase of fluorescence intensity before and after antibody induction was much higher compared to the Affi-PmrB/A system without amplifier.

![ Fluorescence spectrophotometry of Affi-PmrB/A-amplifier system.  ](https://static.igem.wiki/teams/4307/wiki/wiki-result-files/pmr/fig9.png)

**Fig 9. Fluorescence spectrophotometry of Affi-PmrB/A-amplifier system.**
Plasmid **pAffi-PmrB/A-amplifier** was induced with IPTG (final concentration 0.24 mg/mL). The experimental group was then induced with 100 mM human IgG antibody, while the control group did not add any antibody. Fluorescence intensity of EGFP was measured by microplate reader. Averaged results from parallel repetition groups were recorded.

# Affi-NisK/R System

### Plasmid Construction

#### pNisinOE1

##### Plasmid Design

**pNisinOE1** is a pET28a-based plasmid for nisin induced green fluorescence detection carrying:

* NisK and NisR under the control of T7 promoter and lac operator
* inducible promoter PnisA
* EGFP as the reporter gene
* resistance marker KanR for selection

![ Plasmid design of pNisinOE1.  ](https://static.igem.wiki/teams/4307/wiki/wiki-result-files/nisin/fig-10-plasmid-design-of-pnisinoe1.png)

**Fig 10. Plasmid design of pNisinOE1.**

##### Cloning Strategies

NisK, NisR, and PnisA sequences were synthesized according to information on UniProt. PCR with high fidelity Q5 polymerase was conducted to amplify fragments and linearize vectors. Fragments and linearized vectors were assembled by NEBuilder® HiFi DNA assesmbly.

##### Construction Results

* NisK+NisR
* PnisA+EGFP

![ Construction result of pNisinOE1 for nisin-induced fluorescence detection.   ](https://static.igem.wiki/teams/4307/wiki/wiki-result-files/nisin/fig-11-construction-result-of-pnisinoe1-for-nisin-induced-fluorescence-detection.png)

**Fig 11. Construction result of pNisinOE1 for nisin-induced fluorescence detection.**

#### pNisinOE2

##### Plasmid Design

**pNisinOE2** is a nisin induced lacZ expression plasmid derived from pNisinOE1 with the only difference that the EGFP gene is changed into lacZα.

![ Plasmid design of pNisinOE2.   ](https://static.igem.wiki/teams/4307/wiki/wiki-result-files/nisin/fig-12-plasmid-design-of-pnisinoe2.png)

**Fig 12. Plasmid design of pNisinOE2.**

##### Cloning Strategies

lacZ fragment from pUC19 plasmid was amplified by PCR and assembled with PCR linearized pNisinOE1 (left out EGFP gene) using NEBuilder® HiFi DNA assesmbly.

##### Construction Results

* NisK+NisR
* PnisA+lacZ

![ Construction result of pNisinOE2 for nisin-induced lacZ expression.    ](https://static.igem.wiki/teams/4307/wiki/wiki-result-files/nisin/fig-13-construction-result-of-pnisinoe2-for-nisin-induced-lacz-expression.png)

**Fig 13. Construction result of pNisinOE2 for nisin-induced lacZ expression.**

#### pNisin1

##### Plasmid Design

**pNisin1** is a pFB20-based plasmid (gift from Fang Ba) for nisin induced lacZ expression with constitutive NisK and NisR expression, carrying:

* NisK and NisR under the control of constitutive promoter J23100 and his operon terminator
* inducible promoter PnisA
* lacZα as the reporter gene
* resistance marker cmR for selection

![ Plasmid design of pNisin1.    ](https://static.igem.wiki/teams/4307/wiki/wiki-result-files/nisin/fig-14-plasmid-design-of-pnisin1.png)

**Fig 14. Plasmid design of pNisin1.**

##### Cloning Strategies

First, PnisA and lacZα fragments were amplified separately by PCR and HiFi assembled with PCR linearized pFB20 outside J23100 and bacterial terminator, generating an intermediate product.
Then nisK and nisR fragment amplified from pNisinOE1 was assembled with PCR linearized intermediate product outside J23100 and his operon terminator to form final product pNisin1.

##### Construction Results

* lacZ+PnisA+NisK+NisR

![ Construction result of pNisin1 for nisin-induced lacZ expression with constitutive NisK and NisR expression     ](https://static.igem.wiki/teams/4307/wiki/wiki-result-files/nisin/fig-15-construction-result-of-pnisin1-for-nisin-induced-lacz-expression-with-constitutive-nisk-and-nisr-expression.png)

**Fig 15. Construction result of pNisin1 for nisin-induced lacZ expression with constitutive NisK and NisR expression.**

#### pNisin2

##### Plasmid Design

**pNisin2** is our final plasmid for constitutive two-component system expression and nisin induced EGFP expression, derived from **pNisin1** with the only difference that the lacZα gene is changed into EGFP.

![ Plasmid design of pNisin2.    ](https://static.igem.wiki/teams/4307/wiki/wiki-result-files/nisin/fig-16-plasmid-design-of-pnisin2.png)

**Fig 16. Plasmid design of pNisin2.**

##### Cloning Strategies

EGFP fragment from pNisinOE1 was amplified by PCR and assembled with PCR linearized pNisin1 (left out lacZ gene) using NEBuilder® HiFi DNA assesmbly.

##### Construction Results

* EGFP+PnisA+NisK+NisR

![ Construction result of pNisin2 for nisin-induced fluorescence detection with constitutive NisK and NisR expression. ](https://static.igem.wiki/teams/4307/wiki/wiki-result-files/nisin/fig-17-construction-result-of-pnisin2-for-nisin-induced-fluorescence-detection-with-constitutive-nisk-and-nisr-expression-2.png)

**Fig 17. Construction result of pNisin2 for nisin-induced fluorescence detection with constitutive NisK and NisR expression.**

#### pConfocal

##### Plasmid Design

**pConfocal** is a pFB20-based plasmid (gift from Fang Ba) for constitutive NisK-EGFP fusion protein expression carrying:

* NisK and EGFP fusion gene under the control of constitutive promoter J23100 and his operon terminator
* inducible promoter PnisA followed by lacZα (unnecessary for localization observation)
* resistance marker cmR for selection

![ Plasmid design of pConfocal.    ](https://static.igem.wiki/teams/4307/wiki/wiki-result-files/nisin/fig-18-plasmid-design-of-pconfocal.png)

**Fig 18. Plasmid design of pConfocal.**

##### Cloning Strategies

EGFP fragment from pNisinOE1 was amplified by PCR and assembled with PCR linearized pNisin1 (left out nisR gene) using HiFi Assembly.

##### Construction Results

* NisK+EGFP

![ Construction result of pConfocal for constitutive NisK-EGFP localization. ](https://static.igem.wiki/teams/4307/wiki/wiki-result-files/nisin/fig-19-construction-result-of-pconfocal-for-constitutive-nisk-egfp-localization.png)

**Fig 19. Construction result of pConfocal for constitutive NisK-EGFP localization.**

#### pNisAB & pNisC

##### Plasmid Design

**pNisAB** and **pNisC** are both pET-28a based plasmid for NisA-affi production, carrying:

* NisA-affibody with 6xHis tag at C terminal
* NisB and NisC responsible for nisA-affibody modification
* T7 promoter and lac operator. driving the expression of NisA-affibody, NisB, and NisC
* resistance marker KanR or CmR for selection

![ Plasmid design of pNisAB and pNisC.     ](https://static.igem.wiki/teams/4307/wiki/wiki-result-files/nisin/fig-20-plasmid-design-of-pnisab-and-pnisc.png)

**Fig 20. Plasmid design of pNisAB and pNisC.**

##### Cloning Strategies

PCR amplified NisA-affibody gene and NisB gene were assembled with PCR linearized pET28a(kanR). PCR amplified NisC gene was assembled with PCR linearized pET28a(cmR).

##### Construction Results

* NisA-affibody+NisB
* NisC

![ Construction result of pNisAB and pNisC for NisA-affibody expression.       ](https://static.igem.wiki/teams/4307/wiki/wiki-result-files/nisin/fig-21-construction-result-of-pnisab-and-pnisc-for-nisa-affibody-expression.png)

**Fig 21. Construction result of pNisAB and pNisC for NisA-affibody expression.**

### Verification of overexpressed nisin TCS

To test whether nisin TCS can function correctly in *E.coli*, we transformed **pNisinOE1** into *E.coli* strain BL21(DE3) and induced NisK and NisR expression by IPTG. NisR expression was detected by SDS-PAGE (Fig.22). However, we didn’t detect NisK in SDS-PAGE, probably due to its membrane-bound localization.

![ SDS-PAGE result of NisR expression.     ](https://static.igem.wiki/teams/4307/wiki/wiki-result-files/nisin/fig-22-sds-page-result-of-nisr-expression.png)

**Fig 22. SDS-PAGE result of NisR expression.**

Then we tried to induce nisin TCS to function using multiple concentration of nisin. Fluorescence spectrophotometry results by microplate reader demonstrated that the optimal concentration of nisin induction was 1.0 ng/mL (Fig.23). Observation under fluorescence microscope also confirmed that EGFP expression was enhanced when 1.0 ng/ml nisin was added(Fig.24).

![ pNisinOE1 nisin induction results by microplate reader.      ](https://static.igem.wiki/teams/4307/wiki/wiki-result-files/nisin/fig-23-pnisinoe1-nisin-induction-results-by-microplate-reader.png)

**Fig 23.pNisinOE1 nisin induction results by microplate reader.**
Plasmid **pNisinOE1** was induced with multiple concentration of nisin. OD600 was measured to normalize the density of bacteria solution. Fluorescence intensity of EGFP was measured by microplate reader. Averaged results from parallel repetition groups were recorded.

![ pNisinOE1 nisin induction results under fluorescence microscope.       ](https://static.igem.wiki/teams/4307/wiki/wiki-result-files/nisin/fig-24-pnisinoe1-nisin-induction-results-under-fluorescence-microscope.png)

**Fig 24.pNisinOE1 nisin induction results under fluorescence microscope.**
Plasmid **pNisinOE1** was induced with 1.0 ng/mL nisin. Photos were taken under fluorescence microscope (100x).

Noticing that EGFP expression induced by nisin could only be viewed under microplate reader or fluorescence microscope, we tried to use lacZ as a replacement of EGFP to be the reporter gene so as to have a better detection signal visible without microplate reader or microscope, given that lacZ expression can be easily visualized when X-gal is added.

We transformed **pNisinOE2** plasmid into BL21(DE3) and cultured the bacteria on plates with X-gal, IPTG and different concentrations of nisin. However, we ignored the fact that strain BL21(DE3) could express lacZ constitutively, so all the colonies on all the plates were blue(Fig.25). We also used strain DH5α to repeat this experiment. However, probably due to absence of T7 polymerase gene, the induction was not successful as all the colonies on all the plates were white(Fig.26).

![ pNisonOE2 nisin induction results in BL21(DE3).](https://static.igem.wiki/teams/4307/wiki/wiki-result-files/nisin/fig-25-pnisonoe2-nisin-induction-results-in-bl21-de3.png)

**Fig 25.pNisonOE2 nisin induction results in BL21(DE3).**

![ pNisinOE2 nisin induction results in DH5α.      ](https://static.igem.wiki/teams/4307/wiki/wiki-result-files/nisin/fig-26.png)

**Fig 26. pNisinOE2 nisin induction results in DH5α.**

Facing these problems, we decided to constructed lacZ gene knockout BL21(DE3) strain using CRISPR-Cas9 system. We designed four sgRNAs, but unfortunately none of them were effective. Due to time limitation, further knockout experiments were suspended.

### Verification of constitutive nisin TCS

In order to create strains that can express NisK and NisR constitutively without induction by IPTG, we constructed two more nisin induced reporter expression plasmids **pNisin1** and **pNisin2.**

To determine whether the constitutively expressed nisin receptor NisK in our system could locate on cell membrane correctly, we transformed **pConfocal** plasmid into MACH1-T1 and examined it under confocal laser scanning microscope. The imaging result showed stronger fluorescence signal located on bacterial membrane, and fluorescence intensity measured by computer also showed the same distribution(Fig.27), indicating that most NisK-EGFP fusion proteins were located correctly on the membrane.

![ NisK localization under confocal laser scanning microscope. ](https://static.igem.wiki/teams/4307/wiki/wiki-result-files/nisin/fig-27-nisk-localization-under-confocal-laser-scanning-microscope.png)

**Fig 27. NisK localization under confocal laser scanning microscope (100x).**

After confirming the membrane location of NisK, we transformed **pNisin1** plasmid into strain MACH1-T1 and conducted nisin induction assay to obtain a preliminary qualitative result of whether our system could work under constitutive expression. Results showed that the bacteria solution became relatively blue when different concentrations of nisin were added. (Fig.28)

![ pNisin1 nisin induction results.     ](https://static.igem.wiki/teams/4307/wiki/wiki-result-files/nisin/fig-28-pnisin1-nisin-induction-results.png)

**Fig 28. pNisin1 nisin induction results.**

To obtain quantitative results of constitutively expressing NisK and NisR, we transformed **pNisin2** plasmid into MACH1-T1 strain and measured fluorescence intensity of EGFP by microplate reader after nisin induction at different concentration for different induction time(Fig.29). Results showed that 4-hour induction by nisin obtained relatively better result as 0ng/ml nisin group showed lower fluorescence signal. In addition, results from fluorescence microscope observation of 4-hour induction bacteria confirmed successful induction results as induction group showed stronger fluorescence signal(Fig.30).

![ pNisin2 nisin induction results by fluorescence spectrophotometry.](https://static.igem.wiki/teams/4307/wiki/wiki-result-files/nisin/fig-29-pnisin2-nisin-induction-results-by-fluorescence-spectrophotometry.png)

**Fig 29. pNisin2 nisin induction results by fluorescence spectrophotometry.**
Plasmid **pNisin2** was induced with nisin of different concentration for different induction time. OD600 was measured to normalize the density of bacteria solution. Fluorescence intensity of EGFP was measured by microplate reader. Averaged results from parallel repetition groups were recorded.

![ pNisin2 nisin induction results under fluorescence microscope.](https://static.igem.wiki/teams/4307/wiki/wiki-result-files/nisin/fig-30-pnisin2-nisin-induction-results-under-fluorescence-microscope.png)

**Fig 30. pNisin2 nisin induction results under fluorescence microscope (100x).**
Plasmid pNisin2 was induced with 1 ng/mL nisin for 4h. Photos were taken under fluorescence microscope.

To further figure out better nisin concentration for induction, we conducted 4-h induction assay at more detailed nisin concentration(Fig.31), and result showed that 1-2 ng/ml nisin still worked well in constitutive nisin induction system.

![ pNisin2 nisin 4-hour induction results at detailed induction concentration by fluorescence spectrophotometry.](https://static.igem.wiki/teams/4307/wiki/wiki-result-files/nisin/fig-31-pnisin2-nisin-4-hour-induction-results-by-fluorescence-spectrophotometry.png)

**Fig 31. pNisin2 nisin 4-hour induction results at detailed induction concentration by fluorescence spectrophotometry.**
Plasmid pNisin2 was induced with nisin of different concentration. OD600 was measured to normalize the density of bacteria solution. Fluorescence intensity of EGFP was measured by microplate reader. Averaged results from parallel repetition groups were recorded.

We also conducted flow cytometry for 4h induction bacteria to further validate our results(Fig.32). Flow cytometry results also confirmed relatively successful fluorescence induction by nisin and that 1 ng/mL nisin was the optimal induction concentration for 4h induction.

![ pNisin2 nisin induction results by flow cytometry.     ](https://static.igem.wiki/teams/4307/wiki/wiki-result-files/nisin/fig-32-pnisin2-nisin-induction-results-by-flow-cytometry.png)

**Fig 32.pNisin2 nisin induction results by flow cytometry.**

We also invited HUST-China to conduct the induction assay. However, result of nisin induction assay from HUST-China showed different pattern as 0ng/ml nisin group had relatively high fluorescence signal(Fig.33), probably due to the instability of the system.

![ pNisin2 nisin induction results by HUST-China by fluorescence spectrophotometry.      ](https://static.igem.wiki/teams/4307/wiki/wiki-result-files/nisin/fig-33-pnisin2-nisin-induction-results-by-hust-china-by-fluorescence-spectrophotometry.png)

**Fig 33.pNisin2 nisin induction results by HUST-China by fluorescence spectrophotometry.**

Noticing that the result was relatively unstable, and both the fluorescence signals measured by microplate reader and positive bacterial percentage measured by flow cytometry didn’t increase much, which may not fulfill our final goal, we managed to make some improvements to the induction system.

We designed a promoter strength prediction software to perform computer-based directed evolution of enhancing the strength of PnisA promoter. We selected the top 10 mutant promoters generated by the software with the highest predictive promoter strength. The sequences are as follows:

Table 1. Sequences of mutant promoters.

 Promoter | Last 50 bases || PnisA | agtttgttagatacaatgatttcgttcgaaggaactacaaaataaattat |
| PnisRRH01 | agtttgttagatac**t**atga**c**ttcgttcgaagg**t**actacaaaa**ct**aattat |
| PnisRRH02 | agtttgttag**g**tacaatgattt**t**gttc**c**aag**t**aactacaaaataaattat |
| PnisRRH03 | agtttgttagatacaatga**c**ttcgttcg**t**aggaactaca**g**a**c**ta**g**attat |
| PnisRRH04 | agtttgttaga**c**acaatgattt****t****gttcgaag**t**aactacaaaataa**c**ttat |
| PnisRRH05 | agtttgt**g**agatacaatga**c**tt**t**gttcgaag**t**aactacaaaat**c**aattat |
| PnisRRH06 | agtttgttag**t**tacaatga**c**ttcgttc**a**aaggaactacaaaataaa**c**tat |
| PnisRRH07 | agtttgtta**c**atac**t**atga**c**ttcgttc**t**aaggaactacaaaa**c**aaattat |
| PnisRRH08 | agtttgttagatacaatgatttcgttc**a**aag**t**aactacaaa**c**t**g**aattat |
| PnisRRH09 | agtttgttagatacaatga**c**tt**t**gttcgaa**t**gaactacaaaataaa**c**tat |
| PnisRRH10 | agtttgttag**g**tacaatgatttcgttc**a**aaggaactacaaa**c**taa**c**ttat |

For more details on this promoter strength prediction software, please visit [our Software page](https://2022.igem.wiki/tsinghua/software).

We introduced these 10 promoters into our plasmid pNisin2 and transformed them into MACH1-T1 strain to detect EGFP fluorescence signal with and without nisin induction by microplate reader. Results demonstrated that fluorescence signals were enhanced both with and without nisin induction except for PnisRRH07 and PnisRRH10(Fig.34).

![ Fluorescence spectrophotometry results of the 10 mutant promoters with the highest predictive strength.       ](https://static.igem.wiki/teams/4307/wiki/wiki-result-files/nisin/fig-34-fluorescence-spectrophotometry-results-of-the-10-mutant-promoters-with-the-highest-predictive-strength.png)

**Fig 34.Fluorescence spectrophotometry results of the 10 mutant promoters with the highest predictive strength.**

Furthermore, flow cytometry results confirmed that percentages of positive fluorescence signal bacteria also increased both with and without nisin induction(Fig.35), indicating that the improvement of promoter PnisA by our software tool was practical.

![ Flow cytometry results of the 10 mutant promoters with the highest predictive strength.](https://static.igem.wiki/teams/4307/wiki/wiki-result-files/nisin/fig-35-flow-cytometry-results-of-the-10-mutant-promoters-with-the-highest-predictive-strength.png)

**Fig 35.Flow cytometry results of the 10 mutant promoters with the highest predictive strength.**

### Verification of Affi-NisK/R system

The activation of the nisin induced system by EGFR is mediated by our modified fusion protein NisA-affibody ZEGFR:2377. For effective NisA-affibody production, we constructed pNisAB and pNisC, and co-transformed them into BL21(DE3). Nis-affibody was expressed and purified using Ni column. Western blots assay targeting 6xHis tag was conducted to detect nisA-affibody expression(Fig.36). However, the enrichment level of nisA-affibody in elution was relatively low, probably due to unstable binding between the fusion protein and the column.

![ Western blot results to detect NisA-affibody expression.](https://static.igem.wiki/teams/4307/wiki/wiki-result-files/nisin/fig-36-western-blot-results-to-detect-nisa-affibody-expression.png)

**Fig 36.Western blot results to detect NisA-affibody expression.**

Then we tried to induce our constitutive nisin TCS using NisA-affibody that we purified. We conducted induction assay in MACH1-T1 strain containing **pNisin2** plasmid using different volumn of NisA-affibody elution for different time, and found that 4h induction still worked better(Fig.37).

![ Fluorescence spectrophotometry results of pNisin2 induction using NisA-affibody.](https://static.igem.wiki/teams/4307/wiki/wiki-result-files/nisin/fig-37-fluorescence-spectrophotometry-results-of-pnisin2-induction-using-nisa-affibody.png)

**Fig 37.Fluorescence spectrophotometry results of pNisin2 induction using NisA-affibody.**
Plasmid **pNisin2** was induced with NisA-affibody elution of different volumn for different induction time.

Then we conducted 4-h induction assay using detailed volumn of NisA-affibody elution and the elution buffer as the control. Fluorescence spectrophotometry results showed that at some volumn induction by NisA-affibody greatly increased fluorescence signal, demonstrating that our constitutive nisin TCS may be successfully induced by the NisA-affibody that we purified. (Fig.38) Flow cytometry results also confirmed the success of our Affi-NisK/R system. (Fig.39)

![ Fluorescence spectrophotometry results of pNisin2 induction for 4h using NisA-affibody.       ](https://static.igem.wiki/teams/4307/wiki/wiki-result-files/nisin/fig-38-fluorescence-spectrophotometry-results-of-pnisin2-induction-for-4h-using-nisa-affibody.png)

**Fig 38.Fluorescence spectrophotometry results of pNisin2 induction for 4h using NisA-affibody.**
Plasmid **pNisin2** was induced with NisA-affibody elution and elution buffer of different volumn. OD600 was measured to normalize the density of bacteria solution. Fluorescence intensity of EGFP was measured by microplate reader. Averaged results from parallel repetition groups were recorded.

![ Flow cytometry results of pNisin2 induction using NisA-affibody.       ](https://static.igem.wiki/teams/4307/wiki/wiki-result-files/nisin/fig-39-flow-cytometry-results-of-pnisin2-induction-using-nisa-affibody.png)

**Fig 39.Flow cytometry results of pNisin2 induction using NisA-affibody.**

We also invited HUST-China to conduct the induction assay. However, result of NisA-affibody induction assay from HUST-China showed no significant changes of fluorescence signal(figure not shown), probably due to the instability of the system. Thus, further verification experiments of Affi-NisK/R system still need to be carried out in the future.

# Logic Gate

### Plasmid Construction

This section presents the success of our plasmid construction . The cloning strategies and construction results for each plasmid are shown as follows. Detailed potocols can be found on the [Method page](https://2022.igem.wiki/tsinghua/methods).

#### pLogic1 for integrase characterization

##### Plasmid design:

**pLogic1** has the backbone of pET28a, carrying:

* gene sequence to express serine integrase Bxb1
* PBAD/AraC promoter, driving the expression of serine integrase
* OR2-OR1 containing promoter PR flanked by attP and attB sites
* reporter genes to express EGFP and mCherry
* resistance marker AmpR for selection

![Plasmid design of pLogic1 for inergase characterization ](https://static.igem.wiki/teams/4307/wiki/wiki-result-files/logic-gate/plogic1.png)

**Fig 40. Plasmid design of **pLogic1** for inergase characterization.**

##### Cloning strategies

PCR with high fidelity Q5 polymerase was conducted to amplify fragments and linearize vectors. Fragments and linearized vectors were assembled by NEBuilder® HiFi DNA assesmbly. We separated the cloning into two steps for an optimum of efficacy:

* Serine integrase and PBAD/araC were assembled and inserted into pET28a(Carb)
* mCherry-attP-OR-attB-EGFP were assembled and inserted into pET28a(Carb)

##### Construction result

* mCherry-attB-OR-attP-EGFP
* integrase Bxb1

![Construction result of pLogic1 for inergase characterization](https://static.igem.wiki/teams/4307/wiki/wiki-result-files/logic-gate/plogic1-construction-results.png)

**Fig 41. Construction result of pLogic1 for inergase characterization.**

#### pLogic2 for cro/cI characterization

##### Plasmid design

**pLogic2** has the backbone of pET28a, carrying:

* *cro* gene to express Cro protein
* *cI* gene to express cI protein
* PBAD/AraC promoter, driving the expression of cro
* OR2-OR3 containing promoter PRM, driving the expression of cI
* mCherry-attB-OR-attP-EGFP
* resistance marker CmR for selection

![Plasmid design of pLogic2 for cro/cI characterization ](https://static.igem.wiki/teams/4307/wiki/wiki-result-files/logic-gate/plogic2.png)

**Fig 42. Plasmid design of **pLogic2** for inergase characterization.**

##### Cloning Strategies

Fragments were amplified by PCR using the high fidelity Q5 polymerase, whereas vectors were linearized by inverse PCR. Fragments and linearized vectors were assembled by NEBuilder® HiFi DNA assesmbly. We separated the cloning into three steps for an optimum of efficacy:

* cro and PBAD/AraC were assembled and inserted into pET28a(Chl)
* cI and OR2-OR3 were assembled and inserted into pET28a(Chl)
* mCherry-attP-OR-attB-EGFP were assembled and inserted into pET28a(Chl)

##### Construction result

* mCherry-attB-OR-attP-EGFP
* cI
* PBAD/AraC-cro1
* PBAD/AraC-cro2

![Construction result of pLogic2 for cro/cI characterization. ](https://static.igem.wiki/teams/4307/wiki/wiki-result-files/logic-gate/plogic2-construction-results.png)

**Fig 43. Construction result of **pLogic2** for cro/cI characterization.**

### Functional Test for PBAD/AraC System

Since the expression of integrase and cro are under the control of inducible PBAD/AraC system, we first verified the function and intensity of PBAD promoter by constructing verification plasmids that consist of PBAD promoter and a reporter gene to express mCherry, and measuring fluorescence intensity by microplate reader. Given that the effect and intensity of PR promoter within OR1 operator was inconclusive, we compared the promoter intensity with or without OR2-OR1 operator sequence present in the genetic circuit.

Our results proved that the PBAD/AraC system functioned well under induction. The leakage amount before induction was minor. We also proved that the OR2-OR1 operator imposed minor effect on PBAD, so it would not affect our subsequent characterization results of integrase and cro/cI.

![Functional test for PBAD/araC system ](https://static.igem.wiki/teams/4307/wiki/wiki-result-files/logic-gate/pbad-promoter-function-test.png)

**Fig 44. Functional test for PBAD/araC system.**
Plasmids PBAD-mCherry and PBAD-mCherryOR were induced at 37℃ with 1% (w/v) arabinose for 0h, 3h, 6h, and 16h. OD600 was measured to normalize the density of bacteria solution. Fluorescence intensity of mCherry was measured by microplate reader. Averaged results from parallel repetition groups were recorded.

### Characterization of Serine Integrase Function

#### qPCR results

We applied quantitative RT-PCR to measure the inversion efficiency of serine integrase. The primers used in qPCR were designed to amplify sequences in between only after inversion caused by integrase. We analyzed the inversion efficiency of serine integrase by measuring the Ct values of qPCR before and after arabinose induction. The influence of bacterial amount on the Ct values of target gene was blanked by measuring the Ct value of a reference gene (resistance marker gene on the plasmid vector).

After intricate analysis, we summarized valid data and performed regression analysis to test our detection. (Table.2) Even though the exact inversion efficiency cannot be determined, relative magnitude demonstrated that our system induced a two-fold increase in the ratio of inversed promoter sequence. (Fig. 45)

Table 2. qPCR results of serine integrase characterization.

 Induction temperature (℃) | Induction time (h) | Target sequence Ct (unit) | Reference sequence Ct (unit) || Group 1 | Induced | 32.9 | 32.18 |
| Not induced | 35.07 | 31.72 |
| Group 2 | Induced | 14.195 | 14.655 |
| Not induced | 14.91 | 13.615 |
| Group 3 | Induced | 14.905 | 15.28 |
| Not induced | 14.975 | 15.025 |
| Group 4 | Induced | 13.834 | 14.705 |
| Not induced | 14.785 | 13.325 |
| Regression analysis | Induced | - 0.8791 \* p + q - 0.8961 = 0 | |
| Not induced | - 0.9299 \* p + q - 1.5752 = 0 | |

![Characterization of serine integrase inversion efficiency by RT-qPCR. An exemplary qP graph (left) and the regression results (right).](https://static.igem.wiki/teams/4307/wiki/wiki-result-files/logic-gate/logicgatefig45.png)

**Fig 45. Characterization of serine integrase inversion efficiency by RT-qPCR. An exemplary qP graph (left) and the regression results (right).**

The detailed formula deduction and intricate analysis are as follows.

[x]

![Detailed deduction of qP analysis.  ](https://static.igem.wiki/teams/4307/wiki/wiki-result-files/logic-gate/logicgatefig46.png)

**Fig 46. Detailed deduction of qP analysis.**

Click To Know More

#### Fluorescence spectrophotometry results

We applied fluorescence spectrophotometry to measure the fluorescence intensity of the reporter protein by microplate reader to characterize the inversion efficiency of serine integrase. Before inversion, PR promoter within the OR2-OR1 operator drives the expression of EGFP. In contrast, after inversion caused by integrase, PR promoter drives the expression of mCherry.

Our results showed that the fluorescence intensity of mCherry increased significantly after arabinose induction, indicating high inversion efficiency of serine integrase.

![Characterization of serine integrase function by fluorescence spectrophotometry. ](https://static.igem.wiki/teams/4307/wiki/wiki-result-files/logic-gate/integrase-fluorescence-spectrophotometry.png)

**Fig 47. Characterization of serine integrase function by fluorescence spectrophotometry.**
Plasmid **pLogic1** was induced with 1% (w/v) arabinose for 0h, 3h, 6h, and 16h at 37℃. OD600 was measured to normalize the density of bacteria solution. Fluorescence intensity of mCherry was measured by microplate reader. Averaged results from parallel repetition groups were recorded.

### Characterization of cro/cI Function

Plasmid **pLogic2** was designed for characterization of cro/cI function. We applied fluorescence spectrophotometry to measure the fluorescence intensity of reporter protein EGFP by microplate reader.

Our results showed a significant decrease of fluorescence signal before induction when cI was expressed constitutively, indicating that cI associated with OR promoters to repress downstream reporter gene expression. The fluorescence signal increased when cro was expressed under arabinose induction, suggesting that cro successfully blocked the transcription of cI and derepressed the inhibition effect induced by cI. It is worth mentioning that we examined two homologous cro proteins with different sequences that both matched an identical cI protein and received similar results.

![cro_cI_function_veri2 ](https://static.igem.wiki/teams/4307/wiki/wiki-result-files/logic-gate/croci.png)

**Fig 48. Characterization of cro/cI function by fluorescence spectrophotometry.**
Negative control: pET28a(Chl) vector; Postive control: pET28a(Chl) carrying mCherry-attP-OR-attB-EGFP; cI: **pLogic2** w/o induction; cro: **pLogic2** with 1% (w/v) arabinose induction for 16h at 16℃ or 37℃. OD600 was measured to normalize the density of bacteria solution. Fluorescence intensity of EGFP was measured by microplate reader. Averaged results from parallel repetition groups were recorded.

# Reporter System

To enable the engineered bacteria to report assay results in a visulizable way, two color proteins were selected: EiraCFP and mCherry. mCherry is red in natural light; [EiraCFP](http://parts.igem.org/Part%3ABBa_J97000) was added to the part registry in 2014, which is colorless in natural light and blue in UV excitation light. Therefore, mixtures with different ratios of these two proteins will be able to show different degrees of red and blue color in natural light and UV light, respectively, while minimizing the interference between them due to the different excitation light.

We made colorimetric cards by mixing bacteria expressing color proteins in different ratios. This demonstrated the feasibility of selecting these two color proteins as reporters of our detection system.

![Colometric of mCherry (upper panel) and EiraCFP (lower panel).  ](https://static.igem.wiki/teams/4307/wiki/wiki-result-files/reporter-system/reporter.png)

**Fig 49. Colometric of mCherry (upper panel) and EiraCFP (lower panel).**

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