iGEM 2021 — Team BNU-China

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# Team:BNU-China/Notebook

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

### Inspiration

You can see what others teams have done to organize their notes:

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

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# Team:BNU-China/Engineering

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* Human
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  + [Education](https://2021.igem.org/Team%3ABNU-China/Education)
  + [Entrepreneurship](https://2021.igem.org/Team%3ABNU-China/Excellence)
  + [Inclusivity](https://2021.igem.org/Team%3ABNU-China/Inclusivity)
* [Awards](https://2021.igem.org/Team%3ABNU-China/Awards)

Engineering

Success

* [Overview](#1)
* [Anti-mCherry pmrB](#2)
  + [Design](#2.1)
  + [Test](#2.2)
  + [Learn](#2.3)
  + [Improve](#2.4)
  + [Test](#2.5)
* [mRNA switch](#3)
  + [Design](#3.1)
  + [Test](#3.2)
  + [Learn](#3.3)
  + [Improve](#3.4)
  + [Re-learn](#3.5)
  + [Re-design](#3.6)
  + [Further Exploration](#3.7)
* [References](#4)

## Overview

---

Welcome to the engineering success page! Here, we described how we developed and tested the pmrB-anti-mCherry receptor and mRNA switch . During this process of engineering, some of the experiments we planned did not work at the first attempt. Nevertheless, the drawbacks forced us to come up with new ideas and initiated new cycles of engineering, ultimately driving our project forward. On this page we summarized the underlying line of thinking that led to our final engineering success.

## Anti-mCherry pmrB

---

We wanted to design a cheater-proof platform that consists of mutation-reporting workers and specifically-cytotoxic guards to improve the genetic stability. Once the workers transform into cheaters that don’t produce aimed protein, the mutation will be presented and then detected by guards, whose extermination system will therefore be activated and will secrete cytotoxin to selectively target cheaters. One of the most important steps is to recognize the killing signal from guards.

![#](https://static.igem.org/mediawiki/2021/thumb/e/eb/T--BNU-China--es0.jpg/799px-T--BNU-China--es0.jpg)

### Design

There is a pmrBAC system in bacteria, which can be used as a bridge between extracellular signal and intracellular gene expression regulation. Originally, PmrB receptor on cell membrane uses an iron(III)-binding motif to sense the environmental ferric level[[1]](#ref_[1]).Based on the system, we wanted to replace the iron(III)-binding motif with an anti-mCherry motif, which can specifically bind mCherry protein and then facilitate PmrB to phosphorylate PmrA. The PmrC promoter will be activated by phosphorylated PmrA protein. Then it will initiate the expression of CcdB cytotoxin. In a word, mCherry acts as a killing signal to trigger the cheaters' death.

The anti-mCherry single-chain antibody binding region has six binding sites. We replaced the
iron(III)-binding motif with a whole anti-mCherry single-chain antibody sequence, which contained 201bp
in total. Besides, we removed some uncritical sites of anti-mCherry and added a connection
sequence to make sure that the sequence can play a role. The following image is our
preliminary design diagram.(Figure 1)

![#](https://static.igem.org/mediawiki/2021/6/6f/T--BNU-China--engFig1.jpg)

Figure 1 Preliminary design diagram

### Test

###### 1.Construct plasmids

In order to achieve verification, we constructed two plasmids, one was the original design with ccdB(plasmid-B), and the other was using GFP instead of ccdB(plasmid-G) so that we could observe the expression visually. All the fragments were connected to pET-Duet-1 plasmid by enzyme digestion and ligation and transformed into *E. coli* DH5α.

###### 2.Validate plasmids

The products of colony PCR were analyzed by agarose gel electrophoresis and their sizes were verified to be correct. The plasmids were extracted and subjected to single enzyme digestion, and the results of electrophoresis were correct. We performed multiple double enzyme digestion and electrophoresis to verify that the sizes of the products were correct. Then the plasmids were validated by sequencing.

###### 3.Conduct confirmatory experiments

We expanded the culture of bacteria containing plasmid-G(37℃,200rpm)for 12h,divided evenly
into several parts. And we added mCherry solution to one of them and
observed the fluorescence under the fluorescence microscope. However, we did not see our
expected results under a fluorescence microscope.

Then we measured the fluorescence intensity of GFP produced by bacteria with different concentration of mCherry solution added by microplate reader. Not surprisingly, there wasno difference in GFP intensity at different concentrations of mCherry, and the absorbance values of the experimental groups were even similar to the control group.

### Learn

The failure of the experiment led us to rethink our design. We suspected that the length of anti-mCherry sequence was so long that the extracellular polypeptide chain of the modified PmrB failed to form the correct structure, which led to the failure of the verification experiment. We checked the material of previous successful transformations and found that the length of their modified sequence was indeed much shorter than our modified sequence. That's why we finally decided to shorten the length of anti-mCherry sequence as much as possible while retaining its original functions.

### Improve

The anti-mCherry single-chain antibodies binding region had six binding sites. Just as other antibody, anti-mCherry was also axisymmetric. The three binding sites at one side were symmetrical to the three binding sites at the other side. Therefore, we assumed that only three of the binding sites at one side were enough to function properly. [[2]](#ref_[2])After learning from literature, we selected three binding sites from the same side of the antibody:CDR1, CDR2 and CDR3
(Figure 2) [(Part:BBa\_K3805238)](http://parts.igem.org/Part%3ABBa_K3805238), which were only half of the single-chain antibody
binding region to perform its function. We asked Professor Li whether our design scheme was
feasible(Figure 3). He affirmed that our idea was theoretically feasible and worth a try.

![#](https://static.igem.org/mediawiki/2021/thumb/6/66/T--BNU-China--es2.png/736px-T--BNU-China--es2.png)

Figure 2 Optimised design diagram

![#](https://static.igem.org/mediawiki/2021/5/5a/T--BNU-China--engFig3.jpg)

Figure 3 Our communication with professor Li

### Test

Same as before, we conducted two confirmatory experiments after we constructed two plasmids
and validated plasmids.

We examined our results with a fluorescence microscope. Images shot by fluorescence microscope
before and after the addition of mcherry showed results that corresponded to our expectation(Figure 4,5).

![#](https://static.igem.org/mediawiki/2021/8/8b/T--BNU-China--ES-F4.jpg)

Apparently, the higher the concentration of mCherry was, the stronger the fluorescence intensity was, which confirmed that the pathways we constructed properly performed the functions we expected(Figure 6).

![#](https://static.igem.org/mediawiki/2021/f/f8/T--BNU-China--ES-F5.jpg)

Figure 6 Fluorescence intensity produced by cheaters with different concentrations of mCherry

Besides, we proved that the cheaters could accept the mCherry signal to commit suicide(Figure 7).
Through comparison, our results confirmed that the killing efficiency of extermination pathway
reached more than 90%.

![#](https://static.igem.org/mediawiki/2021/thumb/1/12/T--BNU-China--es7.png/800px-T--BNU-China--es7.png)

Figure 7 Comparison of growth of cheaters with and without mCherry
No mCherry was added on the left; mCherry was added on the right

## mRNA switch

---

### Design

mRNA switch is a new part proposed by BNU-China. We designed a pathway with a certain sequence behind its promotor to act as an additional switch to adjust the expression of downstream GFP gene, which represents the extermination system in our project. This unique 12bp mRNA sites in Pathway 2 are complementary with continuous 12bp insulin proteinogen mRNA.[[3]](#ref_[3])[[4]](#ref_[4])

![#](https://static.igem.org/mediawiki/2021/1/19/T--BNU-China--ES-F8.png)

Figure 8 Preliminary design diagram

### Test

At the beginning, in order to test our Pathway 1+Pathway 2 and the expression of insulin proteinogen, we designed a series of bacteria internal（in vivo） experiments to verify the combination of certain 12bp sites normal or changed in insulin proteinogen with complementary sequences in Pathway 2. According to our assumption, it should not be fluorescent when the targeted protein is expressed normally because the specific 12bp sites in insulin proteinogen mRNA will bind to the complementary sequences in Pathway 2. However, if the targeted protein was incorrectly expressed (as in Mutation Ⅰ and Mutation Ⅱ), the cell should show fluorescent. But unfortunately, our results show that all cells could be fluorescence, which didn’t meet our expectations.

![#](https://static.igem.org/mediawiki/2021/2/2a/T--BNU-China--ES-F9-1.png)
![#](https://static.igem.org/mediawiki/2021/1/10/T--BNU-China--ES-F9-2.png)
![#](https://static.igem.org/mediawiki/2021/d/da/T--BNU-China--ES-F9-3.png)

Figure 9 GFP expression (from the left to the right: normal, mutationⅠ, mutationⅡ)

### Learn

We have analyzed the results, discussed the design of experiments, searched for information and asked experts for help. After these, we proposed two possible causes for the unexpected results. (1)The 12bp mRNA binding site is not enough for mRNAs’ interaction. (2)The efficancy of the switch is poorly guaranteed. So, we continued to design a series of experiments to test for our proposed ideas.

![#](https://static.igem.org/mediawiki/2021/c/c6/T--BNU-China--ES-F10.png)

Figure 10 Our group meeting

### Improve

Then, we designed an in vivo experiment to test whether mRNAs can serve as a switch to control the expression of downstream genes. In our experiment, we set three different concentrations for 12bp mRNA in bacterial culture, hoping that the bacteria could internalize the mRNA and indirectly reflect mRNA binding through the expression of GFP. Interestingly, the results showed that the fluorescence intensity of the bacteria with two pathways became lower and lower with the prolongation of incubation time, indicating that the bacteria could absorb short mRNA under normal conditions, and the interaction of mRNA could inhibit the expression level of GFP. 12 bp mRNA can be absorbed by generations of bacterial cells after cell divisions, which causes the ratio of the green fluorescent /cell density (indicated by OD600) to decline continuously over a period of time.

![#](https://static.igem.org/mediawiki/2021/1/1c/T--BNU-China--ES-F11.png)

Figure 11 Optimised design diagram

### Re-learn

However, our team members suggested that the reduced GFP expression in the in vivo experiment might also be due to absorption efficiency caused by strain propagation. After discussion, most of us think that this may exist.

### Re-design

In addition to the in vivo experiment, we also designed a series of mRNA transformation experiments, which is a method that can directly allow mRNA to enter cells, and can record the GFP expression of a specific generation under the constraint of mRNA binding. We used a uniformed 12bp mRNA concentration gradient and the results showed that our mRNA switch is feasible.[[5]](#ref_[5])[[6]](#ref_[6])

![#](https://static.igem.org/mediawiki/2021/d/d2/T--BNU-China--ES-F12.png)

Figure 12 The result of mRNA transformation experiments

### Further Exploration

After demonstrating the feasibility of our switch, we tried to further explore an optimized binding site length. We added a group of 24bp mRNA experiments to compare with the one in the previous experiments. The results showed that gene length, gene concentration and the time after administration and transformation may all have impact on the expression of downstream genes.

![#](https://static.igem.org/mediawiki/2021/2/2f/T--BNU-China--ES-F13.png)

Figure 13 The result of further mRNA transformation experiments

Overall, we have successfully verified the feasibility of our mRNA switch and found several factors that can be adjusted to improve the efficiency of our switch.

## References

---

[1] Liang H, Deng X, Bosscher M, Ji Q, Jensen MP, He C. Engineering bacterial two-component
system PmrA/PmrB to sense lanthanide ions. J Am Chem Soc. 2013 Feb 13;135(6):2037-9.
[2] Mehdi M , Akshay B , Laura G , et al. A Split-Luciferase Reporter Recognizing GFP and mCherry Tags to Facilitate Studies of Protein–Protein Interactions[J]. International Journal of Molecular Sciences, 2017, 18(12):2681-.
[3]Li JH, Liu S, Zhou H, Qu LH, Yang JH. starBase v2.0: decoding miRNA-ceRNA, miRNA-ncRNA and protein-RNA interaction networks from large-scale CLIP-Seq data. Nucleic Acids Res. 2014 Jan;42(Database issue):D92-7. doi: 10.1093/nar/gkt1248. Epub 2013 Dec 1. PMID: 24297251; PMCID: PMC3964941.
[4]Pushparaj PN, Aarthi JJ, Manikandan J, Kumar SD. siRNA, miRNA, and shRNA: in vivo applications. J Dent Res. 2008 Nov;87(11):992-1003. doi: 10.1177/154405910808701109. PMID: 18946005.
[5]Perez Ruiz de Garibay A. Endocytosis in gene therapy with non-viral vectors. Wien Med Wochenschr. 2016 May;166(7-8):227-35. English. doi: 10.1007/s10354-016-0450-5. Epub 2016 May 3. PMID: 27141862.
[6]Kumar P, Nagarajan A, Uchil PD. Electroporation. Cold Spring Harb Protoc. 2019 Jul 1;2019(7). doi: 10.1101/pdb.top096271. PMID: 31262965.

Contact

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# Team:BNU-China/Experiments

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  + [Parts](https://2021.igem.org/Team%3ABNU-China/Parts)
  + [Contribution](https://2021.igem.org/Team%3ABNU-China/Contribution)
  + [Engineering Success](https://2021.igem.org/Team%3ABNU-China/Engineering)
* Human
  + [Human Practices](https://2021.igem.org/Team%3ABNU-China/Human_Practices)
  + [Collaborations](https://2021.igem.org/Team%3ABNU-China/Collaborations)
  + [Partnership](https://2021.igem.org/Team%3ABNU-China/Partnership)
  + [Education](https://2021.igem.org/Team%3ABNU-China/Education)
  + [Entrepreneurship](https://2021.igem.org/Team%3ABNU-China/Excellence)
  + [Inclusivity](https://2021.igem.org/Team%3ABNU-China/Inclusivity)
* [Awards](https://2021.igem.org/Team%3ABNU-China/Awards)

Experiments

## Detailed Protocols

[Close](#return)

### Detailed Protocols:

---

1.Prepare 3 light-proof tubes of 6 mL bacteria solution respectively for the test group and the control group.

2.Observe the 6 samples with fluorescent microscope.

3.Distribute the bacteria solution in each 6 tubes evenly to 3 tubes, which means 18 tubes in total. The three tubes for each sample are named as tube A, B, and C.
4.Culture all the tubes in shaker at 180rpm, 37°C.

5.4h later, take out six A tubes from shaker and centrifugate the tubes at 8000 rpm for 10 minutes. Then separate the supernate and the precipitation. Transfer the supernate to another six light-proof tubes and add 2mL LB medium to suspend bacteria precipitation. Take 200μL from each of the 12 tubes to measure the mCherry fluorescent intensity using microplate reader.

6.Another 40 min later, take out six B tubes from shaker. Repeat step 5.

7.Another 40 min later, take out six C tubes from shaker. Repeat step 5.

* [Present Experiment](#present)
  + [Worker Module](#worker)
  + [Guard Module](#guard)
  + [Killing Pathways and Parts Vertification](#kill)
  + [Cheater-guard Interaction Vertification](#cheat)
  + [Environmental Capacity and Glucose Consumption Measurement](#env)
* [Further Experiment](#further)
  + [Expression Burden Control Circuit](#burden)
  + [Optimal Guard Ratio Vertification](#ratio)

## Present Experiment

---

### Worker Module

###### araC and pBAD promoter expression

**1. Construction of pathways**

We obtain the gene fragments of araC and pBAD promoter by PCR from the plasmid given to us by Sichuan University, and insert the required enzyme digestion sites at both ends. After the completion of PCR, electrophoresis is performed to determine the size of DNA, and then DNA fragments are recovered. Finally, we use the restriction endonucleases for digestion, and then ligate the araC and pBAD promoter fragments into PUC19 plasmids using T4 ligase. Subsequently, in order to verify the inhibitory effect of araC on pBAD promoter, we insert GFP (green fluorescent protein) downstream of pBAD promoter using the same method mentioned above. The intensity of GFP fluoresence represents the transcriptional level of pBAD promoter.

![#](https://static.igem.org/mediawiki/2021/1/11/T--BNU-China--1.1.png)

Figure 1 proinsulin normal 1+3-pathways

![#](https://static.igem.org/mediawiki/2021/1/19/T--BNU-China--2.1.png)

Figure 2 proinsulin mutant 1 1+3-pathways

![#](https://static.igem.org/mediawiki/2021/1/19/T--BNU-China--3.1.png)

Figure 3 proinsulin mutant 2 1+3-pathways

**2.Verification of imported gene pathways**

We transform *E. coli* BL21(DE3) competent cells with the prepared plasmid for overnight culture. Then, we confirm that the constructed plasmid has been successfully transformed *E. coli* Trans 5α by colony PCR as well as PCR using the extracted plasmid as the template. Finally, we preserve the engineered bacteria in glycerol and store them at -80℃ for standby.

**3. Verification of the inhibitory effect of AraC on pBAD promoter**

(1) Inhibitory effect of araC on pBAD promoter

We select *E. coli* BL21(DE3) strain control, proinsulin normal *E. coli* BL21(DE3) strain, proinsulin mutant 1 *E. coli* BL21(DE3) strain and proinsulin mutant 2 *E. coli* BL21(DE3) strain as the four groups of experiment strains. We add 0.6μL 500mM/mL IPTG for each 200μL bacterium to induce the expression of proinsulin and araC fusion protein downstream of the lac operon, then incubate the bacterium at 37℃,200rpm, and the OD600 value and fluorescence intensity of the broth are measured every 30 min.

(2) Fusion expression of araC and proinsulin

We add 60μL 500mM/L IPTG for each 60 mL proinsulin normal strain, proinsulin mutant 1 *E. coli* BL21(DE3) strain and proinsulin mutant 2 *E. coli* BL21(DE3) strain to induce the expression of proinsulin and araC fusion protein downstream of the lac operon for 4h. The three fusion expression proteins are extracted using his-tag protein extraction kit and verified by SDS-PAGE gel.

###### mRNA switch

**1. Construction of pathways**

We synthesize PUC57 plasmids with proinsulin, proinsulin mutant 1, proinsulin mutant 2 genes, and obtain the araC gene by PCR from the plasmid given to us by Sichuan University, then obtain the GFP gene by PCR from the plasmid from our lab. After double digestion with the corresponding enzymes, we use T4 ligase to ligate these gene fragments to the PUC57 plasmids.

![#](https://static.igem.org/mediawiki/2021/6/63/T--BNU-China--E-F4.png)

Figure 4 proinsulin normal 1+2-pathways

![#](https://static.igem.org/mediawiki/2021/7/77/T--BNU-China--E-F5.png)

Figure 5 proinsulin mutant 1 1+2-pathways

![#](https://static.igem.org/mediawiki/2021/e/e2/T--BNU-China--E-F6.png)

Figure 6 proinsulin mutant 2 1+2-pathways

**2. Verification of imported gene pathways**

To verify successful vector construction, we introduce the constructed plasmids into trans 5αcompetent cells, plate and culture overnight. A single colony is picked for colony PCR to determine that both araC and GFP genes have been correctly inserted into the PUC57 plasmids. Finally, we store the engineered bacteria in glycerol for cryopreservation at -80℃.

**3. Feasibility experiments on mRNA switch**

(1) Bacteria internal experiments

We use the *E. coli* BL21(DE3) strain containing a 2-pathway as the control group, and the *E. coli* BL21(DE3) strain containing proinsulin normal strain, proinsulin mutant 1 *E. coli* BL21(DE3) strain and proinsulin mutant 2 *E. coli* BL21(DE3) strain as the experimental group. Each strain is cultured at 200 rpm at 37 ℃ in 5 ml of medium, and the fluorescence is detected under the fluorescence microscope every 1 hour.

(2) Endocytosis experiments

Proinsulin normal *E. coli* BL21(DE3) strain, proinsulin mutant 1 *E. coli* BL21(DE3) strain and proinsulin mutant 2 *E. coli* BL21(DE3) strain are experimental strains. We add 0μL,3μL,5μL,10μL 10μM/L 12bp mRNA fragments complementary to those at the 2-pathway mRNA switch for each 200μL bacterium. The bacterium are cultured at 200rpm at 37 ℃, and the OD600 and fluorescence intensity of the broth are measured every 30 min.

(3）mRNA turnover experiments

In the experiment of RNA natural absorption, we obtain the relevant data that 12bp, 24bp and 36bp length RNA molecules all have an inhibitory effect on fluorescence production. We design RNA systems that inhibit the expression of the downstream kill system of the pathway. Data analysis shows that 12bp RNA is the most inhibitive. In order to exclude the effect of different absorption efficiencies caused by RNA length on the inhibition effect, we design an RNA turnover experiment. The experiment is as follows.

Build engineered bacteria. The treatment of *E. coli* Trans 5αcontaining 1 and 2 pathways with a low temperature pre-cooled CaCl2 solution changed the permeability of its cell membrane and make it a competent cell that could easily absorb exogenous nucleic acid molecules.

Heat shock transformation. We choose two RNA molecules of different lengths (12bp and 24bp in length, respectively) and set a concentration gradient of 0.005 smol/L, 0.01 smol/L, 0.03 smol/μL and 0.05 smol/L for each RNA for heat shock transformation. First of all, the receptor cells are frozen and thawed on the ice, in sterile conditions in accordance with the set concentration gradient to add RNA, gently mixed, incubated on ice bath for 30 minutes, and then incubated in a 42 ℃ metal bath for 90 seconds of static heat. Then centrifuge tubes are rapidly transferred to the ice bath and incubated for 5 minutes. 100 μL of bacteria solution  is taken and added into 600 μL of LB liquid medium and cultured at 37 ℃, 200 rpm.

Data processing and analysis. After transformation, we use microplate reader to measure OD600 and GFP’s expression levels every 5 minutes. After a 60 minutes measurement, we turn to 30 minute measurements, collect the data and model the analysis.

### Guard Module

###### Plasmid Construction

The Guard's gene pathway is mainly composed of Toggle Switch and T7-agrC-agrA pathway. Toggle Switch includes Ptrc-Protein pathway and P2-LacI(LVA)-mCherry pathway. Originally, Ptrc-2 promoter is followed by the gene of target protein. However, to observe the expression of the target protein, we substitute the target protein with GFP, so as to monitor the expression level of the target gene easily.

![#](https://static.igem.org/mediawiki/2021/8/8d/T--BNU-China--7.1.png)

Figure 7 Guard Plasmid

###### OmpT Function Verification

OmpT signal peptide is an excretion tag linked on the N-terminus of protein.We fuse OmpT signal peptide at the N-terminus of mCherry to transport mCherry across outer membrane.
To verify the function of OmpT signal peptide, we place OmpT-mCherry on plasmid pETDuet-1 ,then transform the recombinant plasmid into  *E.coli*  DH5α as the test group. We also construct a recombinant pETDuet-1-mCherry plasmid without OmpT signal peptide and also transform it into  *E.coli*  DH5α as the control group. We use fluorescent microscope and microplate reader to evaluate the transportation efficiency of OmpT signal peptide.

We use the fluorescent microscope to take pictures of the mCherry. We carry out more accurate experiments with microplate reader and carefully analyze the mCherry fluorescent intensity data of the test group and control group. If mCherry can be secreted out of cells with OmpT signal peptide, the fluorescent intensity of the bacteria solution’s supernate after supernation will be distinctly higher than the precipitation compared to the control group. Therefore, we choose the difference of the mCherry fluorescent intensity between the supernate and precipitation as the index of transportation efficiency. The detailed description of this experiment is in the section [“ OmpT Function Verification Protocols”](#open-modal).

###### Promoter Ptrc Examination

To examine the inhibition effect of LacI on promoter Ptrc, we insert GFP gene to the plasmid ptr99a, which contains lacI gene and then is linearized. The linearized product doesn’t contain lacI gene anymore and has homologous hangs on its ends for further self-circulation by in-Fusion cloning. We then obtain two plasmids (one with lacI, the other without) with GFP situated at the downstream of the promoter Ptrc. We then transform *E.coli* DH5α with this two kinds of plasmids respectively and measure the intensity of green fluorescence using fluorescent microscope and microplate reader to verify the inhibition efficiency of lacI on promoter Ptrc.

###### Toggle Switch Verification

To verify the efficacy of our Toggle Switch, we replace our target protein gene with GFP sequence. When a certain amount of exogenous AIP is applied to the bacterial fluid, red fluorescence(mCherry) is observed under the microscope. We then apply a concentration gradient of synthetic AIP to the bacterial fluid to establish an accurate coordination between AIP concentration and mCherry secretion and eventually plot the corresponding curve.

### Killing Pathways and Parts Vertification

When the guard feels AIP, it will secrete mCherry to the outside of the cell, which can activate the cheater's own killing pathway. In this part of the experiment, we aim to verify mCherry's killing operation on cheaters.

###### Obtain fragments

The parts agrB and agrD are obtained from the genome of Staphylococcus aureus ; The pmrC and pmrA are obtained from the plasmid provided by NEU-China; The ccdB and GFP are obtained from the plasmid of our lab.

###### Construct the plasmids

In order to facilitate verification, we construct two plasmids, one is the original design with ccdB(plasmid-B), and the other uses GFP instead of ccdB(plasmid-G).

The obtained fragments are linked to the synthetic anti-mcherry pmrB. Finally, all the fragments are connected to pETDuet-1 plasmid and transformed into *E. coli* DH5α

![#](https://static.igem.org/mediawiki/2021/8/87/T--BNU-China--8.png)
![#](https://static.igem.org/mediawiki/2021/c/c4/T--BNU-China--910.png)

###### Verify the correctness of the plasmids

The PCR products of colony are verified by gel electrophoresis to determine whether the length of the stripsare is correct. The plasmids are extracted and verified by enzyme digestion. Then the fragments are sequenced. The plasmid is transformed into *E. coli* BL21(DE3)(50ul).

###### Conduct confirmatory experiments

Expand the culture of bacteria containing plasmid-G, and then divide evenly into several parts. Add mCherry solution to one of them, and make loading tablets and observe the fluorescence under the fluorescence microscope. Then set the concentration gradient of mCherry solution, and measure the fluorescence intensity of GFP produced by bacteria with different mCherry fluorescence intensity by microplate reader.

Expand the culture of bacteria containing plasmid-B, and then divide evenly into several parts. According to the data measured in the previous experiment, mCherry solution with appropriate concentration is added to several of them and incubated for a period of time. The other is added with buffer as control. Apply the bacteria solution to the solid medium to form single colony. Count the number of growing colonies and calculate the mortality, so as to obtain the killing efficiency.

### Cheater-guard Interaction Vertification

We culture a certain amount of cheaters for a period of time, and then put a certain amount of cheaters’ bacteria solution into the guards’ bacteria solution. Finally, we measure the amount of mCherry produced by the guard with an microplate reader.

### Environmental Capacity and Glucose Consumption Measurement

###### Measure the environmental capacity

The same amount of three bacteria is inoculated in a certain amount of LB liquid medium. Use microplate reader to measure the value of OD600 every 30 minutes. Stop the detection when stability is reached. By modeling, we obtain the value of environmental capacity.

###### Measure glucose consumption

The amount of three kinds of bacteria’s environmental capacity is inoculated into SOC medium, and samples are taken every 5 minutes. After using theD-Glucose Content Assay Kit, the value of OD505 reaction is carried out by microplate reader, and then the absorbance is measured to obtain the concentration of glucose in the sample.

## Further Experiment

---

### Expression Burden Control Circuit

###### Obtain fragments

We synthesize the parts we need: PhtpG1,sgRNA(T7),sgRNA(PBAD),Pconst,dCas9. ﻿

![#](https://static.igem.org/mediawiki/2021/f/f0/T--BNU-China--11.1.jpg)

Figure 11 Gene pathway map of Burden Control Circuit

###### Construct the plasmid

In order to facilitate verification, we construct a new plasmid to accommodate our new parts. Besides, we design a new plasmid only with T7 promoter, promoter and GFP. Finally, all the fragments are connected to pETDuet-1 plasmid and transformed into *E. coli* BL21(DE3).

###### Verify the correctness of the plasmids

The PCR products of colony are verified by gel electrophoresis to determine whether the length of the strip is correct.The plasmids are extracted and verified by enzyme digestion. Then the fragments are sequenced.

###### Conduct confirmatory experiments

A certain number of the bacteria is inoculated in a certain amount of LB medium. Use microplate reader to measure the fluorescence intensity of GFP produced by bacteria every 30 minutes.

### Optimal Guard Ratio Vertification

According to the best guard ratio predicted by the modeling, a mixed experiment of three roles are designed to obtain the expected effect that the production efficiency is improved by using our platform through extracting the target protein. Then we fit with the modeling results to further optimize the hardware design.

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# Team:BNU-China/Results

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* [AraC and pBAD promoter expression part](#1)
  + [Fusion expression of AraC and proinsulin](#1.1)
* [mRNA switch part](#2)
  + [Bacteria internal experiments](#2.1)
  + [Endocytosis experiments](#2.2)
  + [mRNA transformation experiments](#2.3)
* [Guard Module](#3)
  + [Verification of Plasmid Construction](#3.1)
  + [OmpT Function Verification](#3.2)
  + [Promoter PtrcExamination](#3.3)
  + [Toggle Switch Verification](#3.4)
* [Vertification of killing pathways and parts](#4)
* [Verify interaction between the cheater and the guard](#5)
* [Measure the environmental capacity and glucose consumption of three roles (worker, guard and cheater)](#6)

## AraC and pBAD promoter expression part

---

### Fusion expression of AraC and proinsulin

In Pathway 1 + Pathway 3 system, we used a final concentration of 0.5 mmol/mL IPTG to induce expression of the AraC protein in Pathway 1. An increase in the expression of the AraC protein inhibits the pBAD promoter in Pathway 3, resulting in a decrease in the expression of the downstream GFP protein, manifested as a decline in GFP/OD600. Our expectation was that GFP/OD600 for three kinds of E.coli (Normal, Mutation I (which replaces 12 bases of the human proinsulin and AraC fusion protein sequence) and Mutation II (which deletes 8 bases of the human proinsulin and AraC fusion protein sequence )) should all decrease as the induction time increasing. Due to the structural abnormalities of AraC protein caused by the mutations, the AraC inhibition effects of Mutation I and Mutation II should be lower than the Normal one, and the GFP/OD600 should be higher.

In our results, the blank control never showed fluorescence as the induction time increasing, successfully eliminating the interference of unrelated factors. In Normal, Mutation I, and Mutation II, the GFP/OD600 decreased with the prolongation of IPTG induction, which were consistent with our expected results. The GFP/OD600 at different induction times showed Mutation II > Mutation I > Normal, which was also in accordance with our expectation.

![#](https://static.igem.org/mediawiki/2021/thumb/d/de/T--BNU-China--re01.png/800px-T--BNU-China--re01.png)

## mRNA switch part

---

### Bacteria internal experiments

As we expected, the Normal E.coli should not glow fluorescent because the certain 12bp sites in insulin proteinogen combined with complementary sequences in Pathway 2.However, in the case for MutationⅠand MutationⅡ E.coli, it should glow flurescent as for sequences mutation.

But according to our results of bacteria internal experiments, the Normal, MutationⅠand MutationⅡ E.coli all could glow bright fluorescent within 1-12 hours, which means unfortunately mRNA switch had not work.

We proposed two possible reasons for the outcome after discussing with professors and analyzing the results. (1)12bp mRNA bounding site is not enough for mRNAs’ interaction. (2)The efficacy of this switch is poorly guaranteed. We continued to design a series of experiments to test it.

![#](https://static.igem.org/mediawiki/2021/thumb/1/14/T--BNU-China--re02.png/800px-T--BNU-China--re02.png)

### Endocytosis experiments

The results showed that if 12bp mRNA were added to the medium at the concentrations of 0.005μM/μL, 0.01μM/μL and 0.03μM/μL, the fluorescence intensity of bacteria containing the Pathway 1 + Pathway 2 system would become increasingly lower with the incubation time, indicating that bacteria could absorb short mRNA under normal conditions and the mRNA interaction could inhibit the expression level of GFP. We also found that each generation of bacteria could continuously absorb it to maintain an overall continuous decline in GFP/OD600.

![#](https://static.igem.org/mediawiki/2021/thumb/c/cd/T--BNU-China--re03.png/800px-T--BNU-China--re03.png)

### mRNA transformation experiments

We used mRNA transformation assay to artificially transform mRNA into cells and detect fluorescence intensity during the recovery period and the breeding period. As expected, bacteria can emit fluorescence, and its intensity decreases with time. The results showed that the fluorescence intensity per unit volume would decrease in 0-150 minute of the 12bp and 24bp mRNA transformation experiments. This proved that the simple 12bp and 24bp mRNA sequences could bind to certain sites in the mRNA transcribed from Pathway II and serve as switches. It meant that our vision of mRNA switching was possible to some extent.

However, we also found that the efficacy of the mRNA switch did not improve with the increase of mRNA concentration. The possible reasons may lay in the low transformation efficiency or the length of the mRNA binding sites. We will design further experiments to test the optimal length of mRNA binding site in the case of strain self-expression to truly reflect the efficiency of our switch.

![#](https://static.igem.org/mediawiki/2021/thumb/7/75/T--BNU-China--re04.png/800px-T--BNU-China--re04.png)

![#](https://static.igem.org/mediawiki/2021/thumb/0/02/T--BNU-China--re05.png/800px-T--BNU-China--re05.png)

## Guard Module

---

### Verification of Plasmid Construction

We inserted the gene fragment into the plasmid ptr99a in two steps by restriction endonuclease cleavage and ligation. Then we verified the correctness of plasmid construction and product length by colony polymerase chain reaction and gel electrophoresis. The gel electrophoresis pattern is as follows:

![#](https://static.igem.org/mediawiki/2021/thumb/6/6e/T--BNU-China--re06.png/800px-T--BNU-China--re06.png)

### OmpT Function Verification

We inserted the OmpT signal peptide sequence at the upstream of mCherry, the plasmid was verified by colony PCR. The result is as follows:

![#](https://static.igem.org/mediawiki/2021/thumb/5/58/T--BNU-China--re07.png/800px-T--BNU-China--re07.png)

We transformed E.coli DH5α with our validated plasmid and carried out the function verification experiments as we described in the Experiment section. At first, we attempted to take pictures of secreted mCherry with fluorescent microscope. However, the fluorescent images didn’t show much difference between test group and control group. The images are shown below:

![#](https://static.igem.org/mediawiki/2021/7/75/T--BNU-China--4.jpg)

Therefore, we did further exploration and redesigned our verification plan. We carried out more accurate testing experiments with microplate reader and carefully analyzed the mCherry fluorescent intensity data of the test group and the control group.
Here we used the method of paired sample t-test to compare the differences between the two groups of data. And the p-value we got is 1.665e-07 which was far less than 0.01. Therefore, we concluded that there were significant differences between the two groups of data, which successfully confirmed the OmpT guidance on mCherry extracellular secretion. The data of fluorescent intensity measured by microplate reader is as follows:

![#](https://static.igem.org/mediawiki/2021/5/53/T--BNU-China--figTemp5.jpg)

### Promoter Ptrc Examination

We obtained two plasmids in which the green fluorescent protein was located at downstream of the promoter Ptrc. The difference between them is whether they contains lacI. To verify the construction of the two plasmids, we adopted colony PCR,and the gel electrophoresis profiles of the plasmid with or without lacI are as follows:

![#](https://static.igem.org/mediawiki/2021/thumb/0/06/T--BNU-China--re10.png/800px-T--BNU-China--re10.png)

We then transformed E.coli DH5α with the two kinds of plasmids respectively and measured the intensity of green fluorescence with fluorescence microscope and microplate reader. E.coli with lacI expression shows significantly less green fluorescent density, which proved that lacI can inhibit the downstream transcription of Ptrc.
Below is the images observed under fluorescence microscope with or without lacI.

![#](https://static.igem.org/mediawiki/2021/thumb/0/0e/T--BNU-China--re11.png/800px-T--BNU-China--re11.png)

### Toggle Switch Verification

When a certain amount of synthetic AIP was applied to the bacterial solution, red fluorescence was observed under the fluorescence microscope(Figure12.A). After a period of time, red fluorescence disappeared, and green fluorescence was observed under the fluorescence microscope(Figure 12.B).

![#](https://static.igem.org/mediawiki/2021/d/d1/T--BNU-China--1215.jpg)

We then applied a concentration gradient of synthetic AIP to the bacterial fluid to establish an accurate coordination between AIP concentration and mCherry secretion and plotted the corresponding curve.

The corresponding curve is shown below:

![#](https://static.igem.org/mediawiki/2021/thumb/0/07/T--BNU-China--re13.png/800px-T--BNU-China--re13.png)

## Verification of killing pathways and parts

---

When guards perceive AIP signals, they would secrete mCherry outside of the cell, which could trigger the cheater killing pathway. In this part of the experiment, we were intended to verify the mCherry's killing effect on cheaters.

1.Verify the correctness of the plasmids:

The PCR products of bacterial colonies were verified by gel electrophoresis to determine whether the strip length was correct. Plasmids were extracted and verified by enzyme digestion. The fragment are then sequenced.
The gel electrophoresis bands of the two plasmids were as follows:

![#](https://static.igem.org/mediawiki/2021/thumb/a/ab/T--BNU-China--re1415.png/559px-T--BNU-China--re1415.png)

Through the above two gel electrophoresis stripes, we can see that the stripes are correct, which means that our plasmids are correct as well.

2.Conduct confirmatory experiments:

We expanded the culture of bacteria containing plasmid-G, and divided these bacteria into several groups. Then we added mCherry protein solution to one of them, made mountings and observed the fluorescence using the fluorescence microscope. Subsequently, we set the concentration gradient of mCherry protein solution, and measured the fluorescence intensity of GFP under different mCherry protein concentration using microplate reader.

Figures under fluorescence microscope before and after adding mCherry protein solution are as follows:

![#](https://static.igem.org/mediawiki/2021/thumb/c/c6/T--BNU-China--re1617.png/800px-T--BNU-China--re1617.png)

The GFP production represented the switching on of the cheaters’ killing pathways. mCherry conglobation leads to a positive feedback regulation. Once cheaters bind to one mCherry protein molecule, they tend to bind more . And the result is consistent with the prediction of our modeling.

The fluorescence intensity of GFP under different concentrations of mCherry protein solution is shown below:

![#](https://static.igem.org/mediawiki/2021/4/44/T--BNU-China--re18.png)

The fitting curve showed that the expression of GFP may improve with the increase of mCherry. This proves that the intensity of the killing pathways may be enhanced when the concentration of mCherry protein grew higher.

We expanded the culture of bacteria containing plasmid-B, and divided these bacteria into several groups. According to the data measured in the previous experiment, mCherry protein solution of appropriate concentration was added to several groups of bacteria and incubated for a period of time. The remain was added with buffer as control. Afterwards, we applied the culture medium to the solid medium to form single colony. Then we counted the number of growing colonies and calculate the mortality, so as to obtain the killing efficiency.

The colony growth after adding mCherry or buffer solution are as follows:

![#](https://static.igem.org/mediawiki/2021/thumb/9/92/T--BNU-China--re19.png/800px-T--BNU-China--re19.png)

Through spreading plate verification, we found that most cheaters died in our system, which indicated that our platform possesses high killing efficiency. Additionally,, we find that our killing efficiency has reached 90% through further calculation.

## Verify interaction between the cheater and the guard

---

We cultured a certain amount of cheaters for a period of time, and then put a certain amount of cheaters’ culture medium into the guard's culture medium. Finally, we measured the concentration of mCherry protein produced by the guard with a microplate reader.

The concentration of mCherry protein produced by guards is shown below:

![#](https://static.igem.org/mediawiki/2021/thumb/e/e4/T--BNU-China--re20.png/800px-T--BNU-China--re20.png)

According to the above figure, we can see that cheaters can produce AIP, which will stimulate guards to produce mCherry protein subsequently.

## Measure the environmental capacity and glucose consumption of three roles (worker, guard and cheater)

---

1.Measure the environmental capacity：
The same amount of three bacteria was inoculated in a certain amount of LB medium. Then we used microplate reader to measure the value of OD600 every 30 minutes. We continued the detection until stability has been reached. Through modeling, we obtained the value of environmental capacity.

The fitting curves are as follows:

![#](https://static.igem.org/mediawiki/2021/thumb/c/cf/T--BNU-China--re21.png/800px-T--BNU-China--re21.png)

2.Measure glucose consumption：
The environmental capacity of the three bacteria was inoculated into SOC culture medium, and samples were taken once every 5 minutes. After the D-glucose content determination kit was used, the OD505 reaction value was performed with a microplate reader, and the absorbance was measured to obtain the glucose concentration in the sample.
The sugar consumption table is shown below:

![#](https://static.igem.org/mediawiki/2021/thumb/3/3e/T--BNU-China--re22.png/800px-T--BNU-China--re22.png)

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