iGEM 2022 — Team bnu-china

{% 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 %}

## What should this page contain?

---

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

## Inspirations

---

* [2018 Munich](http://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)

{% endblock %}

![](https://static.igem.wiki/teams/4117/wiki/images/loading/rocket.gif)

l
o
a
d
i
n
g
.
.
.

# Engineering

#####

* [**Abstract**](#first)
* [**THC-sensing**](#second)
* [**THC metabolism**](#third)
* [**Reference**](#forth)

# Abstract

An engineering cycle should be established from the beginning and undergoes continuous improvement with the results of experiments, thus, giving birth to a successful engineered product. And that is why and how we document our engineering below.

In the first stage, we reviewed the literature to summarize the metabolism of THC, and tentatively determined that we would engineer *E.coli* Nissle 1917 to express a human-derived degradative enzyme line to transfer Δ9-THC into non-psychoactive substance. After we assessed the difficulty of directly constructing a plasmid containing three enzymes simultaneously, we decided to seperate them into three vectors to obtain a higher operability in our experiments, but still under the sensing of Δ9-THC.

# THC-sensing module

### Imagine 1: What can we use to receive the signal?

At the very beginning, we came up with using G-protein-coupled receptor called CB1 that can sense the Δ9-THC and its analogues. After a series of signal transduction, the adenylate cyclase is activated and cAMP is produced. Thus, we planned to find a kind of promoter that can be activated by cAMP to reach the visualization of the sensing of Δ9-THC by expressing special proteins. However, we then found the method uncontrollable since there are other ways that can influence the content of cAMP in the cell. Besides, we could not find the suitable promoter which can accurately and specifically sense the cAMP.

It was then that the sensing system of 2021 BNU-China popped into our mind. Last year, they replaced the iron(III)-binding motif of Pmr system with an anti-mCherry motif and successfully achieved sensing mcherry and coding proteins. Considering that they have already proved the feasibility of the system, we decided to modify their design.

### Design 1: How can we sense the THC?

The main focus of our design is on sensing the Δ9-THC. PmrB receptors of the Pmr system, which is located in the cell membrane, use the iron (III) -binding motif to sense iron levels in the physiological environment . We replaced the iron (III) binding motif with an anti-THC motif(2019 Queens\_Canada), which specifically binds Δ9-THC and then promotes PmrB to phosphorylate PmrA protein. Phosphorylated PmrA protein further activates the PmrC promoter, initiating the expression of reporter protein and enzymes to degrade THC.

### Imagine 2: How can we visualize the sensing results?

After sensing the Δ9-THC by the Pmr system, we tried to find a kind of reporter protein to distinctly remind the people who have taken in Δ9-THC by mistake.

At the very beginning, we considered the most common reporter protein--GFP. We imagined users testing their excrement to know if they have any mis-ingestion of cannabis. Soon we found that testing fluorescence is not convenient. Luckily, a piece of news inspired us--Eating edible coloring pigments results in colorful excrement. Because of that, we chose to express a kind of workable edible coloring pigment to visualize the results.

### Design 2: What can we use to achieve visualization?

We decided to make our engineering bacteria secrete C-phycocyanin to verify if they have successfully finished the sensing pathway . C-phycocyanin is a natural blue pigment extracted from Spirulina. It is one of the rare pigment proteins in nature, which allows us to make a distinction between daily food and cannabis intake. It is not only bright in color, but also a harmless food.

![](https://static.igem.wiki/teams/4117/wiki/images/engineering-1.png)

Fig.1 Schematic representation of the α (brighter color) and β (darker color) subunits of phycocyanin[[1]](#ref-0).

### Build 1: How can we build it?

We got the full sequence of PmrA, PmrB, PmrC and C-phycocyanin from NCBI. While replacing the original iron (III) -binding motif of PmrB, we put C-phycocyanin downstream of the PmrC promoter and connected all of them with plasmid PUC57. We also added His-tag linked with the C-terminal of the C-phycocyanin for purification and OmpA signal sequence with the C-terminal of the C-phycocyanin for extracellular secretion.

### Test 1

To confirm the correct structure and expression of C-phycocyanin, we linked phycocyanin to lacO which can be activated by IPTG in plasmid PET-Duet1. We transfected PET-Duet1 into *E.coli*(DH5α) for amplification. Then we extracted the plasmid for sequencing. The result met our expectations generally. So we transfected the plasmids into *E.coli*(BL21) to express the C-phycocyanin. After inducing with IPTG, we centrifuged and broke the bacteria to acquire the lysate. Finally we used Nickel column to adsorb C-phycocyanin and verified it.
Following that, we began to verify our pathway by using 11-OH-Δ9-THC instead of the controlled drug △9-THC. We induced our engineering bacteria by adding 11-OH-△9-THC. The experiment is still on going……

### Learn & Improve 1

During our experiment, we surprisingly found that the C-phycocyanin might be oxidized to brown but not blue in the air. [[1]](#ref-0)This may cause interference for visualization. Therefore, we tried to find some feasible methods to ensure the stability of C-phycocyanin. Finally, the whey protein came into our sights, which was proved to result in blue color maintenance.[[2]](#ref-1) We designed to pack the whey protein powder and our engineering bacteria in a kind of suitable capsule, so as to release them together and protect the C-phycocyanin. Besides, if this way cannot work well, we will turn back to fluorescin like mCherry and improve the hardware.

![](https://static.igem.wiki/teams/4117/wiki/images/engineering-2.png)

Fig.2 We tried to find some feasible methods to ensure the stability of C-phycocyanin.

# THC metabolism module

### Imagine 1: How to develop a THC-metabolizing pathway that works upon sensing Δ-9-THC?

The focus of our design is to help people degrade addictive cannabis that they have ingested in their intestine in a safe way, which means the addictive substance is converted to non-addictive substance. In human body, Δ9-THC is hydroxylated to 11-OH-Δ9-THC, which is still psychoactive and is further oxidized to psychoactive Δ9-THC-COOH. In the course of our in-depth review of the literature, we introduced three enzymes, CYP2C9, CYP2C19[[3]](#ref-2)[[4]](#ref-3), which are necessary for the metabolism of Δ9-THC , and UGT1A3[[5]](#ref-4), which converts Δ9-THC-COOH into non-addictive cannabinoid glucuronic acid.

![](https://static.igem.wiki/teams/4117/wiki/images/engineering-3.png)

Fig.3 Cytochrome P-450(CYP-450) metabolic pathways for cannabinoids and investigated metabolites based on in vitro data.[[4]](#ref-3)Supporting data (Bland et al., 2005; Bornheim et al., 1992; Chimalakonda et al., 2012; Jiang et al., 2011; Matsunaga et al., 2000; Richardson et al.,1995; Watanabe et al., 1995, 2002, 2007).

![](https://static.igem.wiki/teams/4117/wiki/images/engineering-4.png)

Fig.4 Glucuronidation activity screening. Selected recombinant UGT isoforms and human liver microsomes were screened for activity toward CBN, THC-OH, THC-COOH, and CBD. Glucuronidation activities were measured by incubating microsomal protein 5 μg of recombinant UGT; 50 μg of human liver (HL)] with 500 μM substrate and 4 mM UDP-GlcUA. All reactions were normalized as described under Materials and Methods.[[5]](#ref-4)

### Design 1: How do we ensure the Δ9-THC conversion in the body?

At first, we hoped to introduce the above pathway into the intestinal flora to metabolize Δ-9-THC by expressing three enzymes, so as to avoid the harm and influence caused by ingestion of cannabis. However, in the actual construction of plasmids, we found that the introduction of three enzyme genes into a plasmid would make the plasmid too large, and CYP2C9 and CYP2C19 have homologous sequences, which would make the construction of plasmids difficult. Therefore, we decided to introduce the gene sequences of the three enzymes into three plasmids respectively, and all of them were induced and detected by PET-DUET as the chassis.

### Build 1: How can we build it?

We obtained the sequences of CYP2C9, CYP2C19 and UGT1A3 from NCBI. We designed the sequence of these enzymes by codon optimization and added the OmpT signal peptide sequence with the N-terminal of each gene sequence for surface display. We introduced these three enzyme genes into three plasmids separately.

### Test 1: Do we get what we want?

The results generally met our expectations. After culturing *E.coli* for 5h in a shaking bed, we took ultrasonic fragmentation to break the cells, purified the protein through his-tag protein extraction kit and verified it by SDS-PAGE. We finally obtained the correct bands.

In subsequent experiments, we will use Western Blot to detect protein expression. Moreover, we found Enzyme immunoassay for human cytochrome P450 family member 2C19 (CYP2C19) ,Enzyme immunoassay for human cytochrome P450 family member 2C9 (CYP2C9)kit and Human uridine diphosphate glucuronosyltransferase (UGT) enzyme immunoassay kit to verify the biological activity of three enzymes.

### Learn & Improve 1

For the idea that we express three enzymes separately, we were questioned in subsequent communication with professors and other teams - how to adjust the proportion of the three bacteria carrying the enzyme separately? Is there an interaction between them? These talks really inspired us that in future improvements, we would consider emerging technologies like the Crispr-Cas9. We hope to express sgRNA and Cas9 through a plasmid, perform fusion PCR on the genes of the three enzymes to construct Donor DNA, transfer the plasmid substituted with sgRNA and Cas9 and Donor DNA into *Escherichia coli* at the same time, and guide Cas9 protein through the targeted localization of sgRNA. Hopefully the genes for three enzymes will be inserted into the genome of *E.coli*.

(1)The host strain is prepared into competent cells, and λ-Red recombination system is integrated in the genome of the host strain in advance to induce the expression of recombinase during the preparation of competent cells;

(2)Plasmid pCas9cur, plasmid psgRNA and linear Donor DNA are co-transferred into host cells, or plasmid psgRNA and linear Donor DNA are co-transferred into host cells containing plasmid pCas9cur;

(3)Linear Donor DNA recombines with genome target sequences mediated by recombinase, and the constitutively expressed Cas9 and sgRNA form a complex to recognize and cut the genome sequences that have not been recombined.

![](https://static.igem.wiki/teams/4117/wiki/images/engineering-5.png)

Fig.5 We will take the Crispr-Cas9 technology to solve the problems of expression of three enzymes.

# References

- Pez Jaeschke D, Rocha Teixeira I, Damasceno Ferreira Marczak L, Domeneghini Mercali G. Phycocyanin from Spirulina: A review of extraction methods and stability. Food Res Int. 2021 May;143:110314. doi: 10.1016/j.foodres.2021.110314. Epub 2021 Mar 17. PMID: 33992333.
  [Full text](https://pubmed.ncbi.nlm.nih.gov/33992333/)
  [Back to text](#intext1)

- Zhang S, Zhang Z, Dadmohammadi Y, Li Y, Jaiswal A, Abbaspourrad A. Whey protein improves the stability of C-phycocyanin in acidified conditions during light storage. Food Chem. 2021 May 15;344:128642. doi: 10.1016/j.foodchem.2020.128642. Epub 2020 Nov 16. PMID: 33223303.
  [Full text](https://pubmed.ncbi.nlm.nih.gov/33223303/)
  [Back to text](#intext2)

- Watanabe K, Yamaori S, Funahashi T, Kimura T, Yamamoto I. Cytochrome P450 enzymes involved in the metabolism of tetrahydrocannabinols and cannabinol by human hepatic microsomes. Life Sci. 2007 Mar 20;80(15):1415-9. doi: 10.1016/j.lfs.2006.12.032. Epub 2007 Jan 17. PMID: 17303175.
  [Full text](https://pubmed.ncbi.nlm.nih.gov/17303175/)
  [Back to text](#intext3)

- Stout SM, Cimino NM. Exogenous cannabinoids as substrates, inhibitors, and inducers of human drug metabolizing enzymes: a systematic review. Drug Metab Rev. 2014 Feb;46(1):86-95. doi: 10.3109/03602532.2013.849268. Epub 2013 Oct 25. PMID: 24160757.
  [Full text](https://pubmed.ncbi.nlm.nih.gov/24160757/)
  [Back to text](#intext4)

- Mazur A, Lichti CF, Prather PL, Zielinska AK, Bratton SM, Gallus-Zawada A, Finel M, Miller GP, Radomińska-Pandya A, Moran JH. Characterization of human hepatic and extrahepatic UDP-glucuronosyltransferase enzymes involved in the metabolism of classic cannabinoids. Drug Metab Dispos. 2009 Jul;37(7):1496-504. doi: 10.1124/dmd.109.026898. Epub 2009 Apr 1. PMID: 19339377; PMCID: PMC2698943
  [Full text](https://pubmed.ncbi.nlm.nih.gov/19339377/)
  [Back to text](#intext5)

![](https://static.igem.wiki/teams/4117/wiki/images/toppic.png)

{% include 'pages/new\_footer.html' %}

{% 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 %}

![](https://static.igem.wiki/teams/4117/wiki/images/loading/rocket.gif)

l
o
a
d
i
n
g
.
.
.

# Result

#####

* [**Overview**](#first)
* [**THC-sensing**](#second)
* [**ΔTHC metabolism**](#third)
* [**Suicide Module**](#forth)

# Overview

The results of all our design will be shown in this page. So far we had finished parts of our scheduled experiments and the results had proved the reliability and feasibility of our design to some extent. However, some picture may be not that beautiful because of the lack of time, and we are still conducting experiments for a better results. Besides, the results of verification of Δ9-THC-sensing system and the activity of the enzymes will soon be added!!!

# THC-sensing module

### C-Phycocyanin extracellular secretion under IPTG induction

##### Verification of Plasmid Construction

We inserted the gene fragment into the plasmid PET-Duet1 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.wiki/teams/4117/wiki/images/result/1.png)

Fig.1 Gel electrophoresis results of the colony PCR of the plasmid Lane M: Marker III; Lane 1-2: colony PCR products of the T7-C-Phycocyanin plasmid(1237bp)

![](https://static.igem.wiki/teams/4117/wiki/images/result/2.png)

Fig. 2 pET-Duet1- pInteraction-IPTG sequencing map

##### C-Phycocyanin Purification Verification

We transformed *E.coli* BL21(DE3) 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 C-Phycocyanin with the optical microscope, however, the images didn’t show much difference between test group and control group and couldn’t show the blue. We guessed that the amount of protein exocytosis was too small to be seen by the optical microscope and we chose to use SDS-PAGE gel to verify the presence of protein.

![](https://static.igem.wiki/teams/4117/wiki/images/result/resultfig3new.png)

Fig.3 SDS-PAGE map of C-Phycocyanin alpha subunit secreted extracellularly by *E.coli*
Lane M1&Lane M2: Premixed Protein Marker(Broad); Lane 1-2: IPTG induced positive transformant; Lane 3-4: IPTG induced positive transformant passing through his-tag protein extraction kit. (C-Phycocyanin alpha subunit is approximately 20.6kD)

### C-Phycocyanin extracellular secretion under Δ9-THC induction

##### C-Phycocyanin Purification Verification

Δ9-THC is banned for private sale in China, we chose the non-psychoactive 11-OH-Δ9-THC to test the normal function of the sensing protein. However, the 11-OH-Δ9-THC is soluble in methanol for transport, it was not until recently that we isolated 11-OH-Δ9-THC from methanol. We have planned Δ9-THC induction experiments (see our experiment) and are working hard to carry out these experiments. If there are subsequent experimental results, we will update our wiki!

# ΔTHC metabolism module

### Verification of Δ9-THC Metabolism Pathway and Parts

We construct the Δ9-THC metabolism pathway of cyp2c9, cyp2c19 and ugt1a3 which induced by Δ9-THC.

##### Verification of plasmids Construction

We insert the gene fragments of cyp2c9/cyp2c19/ugt1a3, ompT and His-tag into the pET-Duet1 downstream of the t7 promoter and upstream of the t7 terminator, and verify the bacterial colony PCR products by gel electrophoresis to initially determine the correctness of the plasmid construction. The results of gel electrophoresis bands of the plasmids are shown in Fig.4- 6:

![](https://static.igem.wiki/teams/4117/wiki/images/result/4.png)

Fig. 4 Gel electrophoresis results of cyp2c9 (100% positive rate)

![](https://static.igem.wiki/teams/4117/wiki/images/result/5.png)

Fig. 5 Gel electrophoresis results of cyp2c19 (87.5% positive rate)

![](https://static.igem.wiki/teams/4117/wiki/images/result/6.png)

Fig. 6 Gel electrophoresis results of ugt1a3 colony PCR (100% positive rate)

After that we extract the plasmids and verify the correct construction of the plasmids by sequencing them by the company. The plasmid profiles returned by the company are shown in Fig.7-9.

![](https://static.igem.wiki/teams/4117/wiki/images/result/7.png)

Fig.7 pET-Duet1- cyp2c9 sequencing map

![](https://static.igem.wiki/teams/4117/wiki/images/result/8.png)

Fig.8 pET-Duet1- cyp2c19 sequencing map

![](https://static.igem.wiki/teams/4117/wiki/images/result/9.png)

Fig.9 pET-Duet1-ugt1a3 sequencing map

### Protein Expression, Purification Verification

The fusion expression proteins are purified by His-Tag protein extraction kit and verified by SDS-PAGE gel. The results of the gradient elution SDS-PAGE of the three target proteins are shown in Figures 6-8. The results show that the proteins are successfully expressed.

![](https://static.igem.wiki/teams/4117/wiki/images/result/10.png)

Fig.10 The gradient elution SDS-PAGE results of CYP2C9 (the target protein bands are in the red box)

![](https://static.igem.wiki/teams/4117/wiki/images/result/11.png)

Fig.11 SDS-PAGE results of gradient elution of CYP2C19 (the target protein bands are in the red box)

![](https://static.igem.wiki/teams/4117/wiki/images/result/12.png)

Fig.12 SDS-PAGE results of gradient elution of UGT1A3 (target protein bands in red box)

# Suicide Module

We use two safety modules to keep the engineered bacteria under restriction. First, arabinose suicide induction module allows users to terminate the product at any time. Second, the use of 37℃ temperature control suicide switch prevents engineering bacteria strain from polluting the environment outside human body.

### Verification of Plasmid Constructione

To verify the RNA thermometer, we replaced mazE and mazF following temperature controlled RBS fragment with GFP on the plasmid.

Bacterial colonies PCR products were verified to be correct by gel electrophoresis. The gel electrophoresis bands of the plasmid were as Fig.13:

![](https://static.igem.wiki/teams/4117/wiki/images/result/13.png)

Fig.13 Gel electrophoresis results of colony PCR (Lane10:RNA Thermometer Verification,5119bp)

After that we extract the plasmids and verify the correct construction of the plasmids by sequencing them by the company. The plasmid profiles returned by the company are shown in Fig.14.

![](https://static.igem.wiki/teams/4117/wiki/images/result/14.png)

Fig.14 Results of plasmid RNA Thermometer Verification sequencing

### Conduct confirmatory experiments

##### Arabinose-induced kill switch

L-arabinose is commonly used to induce the engineered bacteria to stop proliferation. It is also a way to prevent engineered bacteria from spreading and leaking into the soil, because there is enough arabinose in the soil. We used five different concentrations of arabinose (0%, 0.05%, 0.1%, 0.15%, 0.2%) in the experiment to induce the engineered bacteria. Samples were taken every hour to measure the OD600 to get the total number of unruptured bacteria. The killing effect was observed by counting the viable cells by spreading diluted samples on plates. The data of OD600 measurement are shown in Fig.15 and the growth of colonies is shown in Fig.16.

![](https://static.igem.wiki/teams/4117/wiki/images/result/15.png)

Fig.15 The growth of engineered bacteria in different arabinose concentration

![](https://static.igem.wiki/teams/4117/wiki/images/result/16.png)

Fig.16 Comparison of growth of the engineered bacteria treated with L-arabinose at different concentrations and time duration. (from left to the right: 1h, 2h, 3h, 4h; top to bottom: 0%, 0.05%, 0.1%, 0.15%, 0.2%)

![](https://static.igem.wiki/teams/4117/wiki/images/result/17.png)

Fig.17 The numbers of colonies after incubating in different arabinose concentrations

Through spreading plate verification(Fig.18) combined with modeling results(Fig.19), most of the engineered bacteria die at 2749s which means that the system has the high killing efficiency. And further calculation showed that the optimum killing concentration is 0.1%.

![](https://static.igem.wiki/teams/4117/wiki/images/result/18.png)

Fig.18 Comparison of growth of the engineered bacteria treated with 0.1% L-arabinose at different time duration, the dilution of the bacterial solution is also different. (A) 0.5h, 106 (B) 0.75h, 105 (C) 1h,105 (D) 1.5h, 105 (E) 2h, 105 (F) 2.5h, 105 (G) 3h,105 (H) 3.5h,104 (I) 4h, 10^3

![](https://static.igem.wiki/teams/4117/wiki/images/result/19.png)

Fig.19 The numbers of remaining live cells incubated in 0.1% arabinose LB medium over time.

##### Cold-triggered toxin suicide switch

In order to prevent the engineered bacteria from polluting the environment outside human body, the 37℃ temperature control suicide switch was introduced, and the toxin-antitoxin system was used to realize the suicide of engineered bacteria in the non-37 ℃ environment. The strain was incubated at 37 ℃ and then divided equally into two conical flasks. Two conical flasks were incubated at 28℃ and 37℃ for shock culture. Samples were taken hourly for OD600 measurements to get the total number of unruptured bacteria, and the killing effect was observed by counting viable cells by spreading diluted samples on plates. The data of OD600 measurement are shown in Fig.20 and the expression of mazE is shown in Fig.21.

![](https://static.igem.wiki/teams/4117/wiki/images/result/20.png)

Fig.20 The data of OD600 measurement.

![](https://static.igem.wiki/teams/4117/wiki/images/result/21.png)

Fig.21 The expression of mazE, mazF and mazEF at different temperatures and different incubation time.

On the left side is the situation where temperature ≥ 37 ℃, and concentration of mazF, the toxin, remains at single digit throughout the 2-hour simulation, which is way lower than the cleavage threshold. While the simulation result of ≤37 ℃ on the right side implies that mazF can be produced at a pretty fast rate, its degradation rate is slow enough to be ignored, and there is not much mazE that binds to it. Thus, it can eventually hold a high concentration to be toxin to cells. In this case, the concentration of the antitoxin mazE peaks around the first few tens of seconds and then declines slowly until 2 hours later. Hence it's safe to draw the conclusion that the bacteria can be eliminated for some degree when leakage happens. We will further explain how we developed the suicide module and explore the probable principle for low killing efficiency.

Reflection：We constructed a toxin-antitoxin system controlled by RBS thermometer, and the experimental results showed that the killing effect of RBS thermometer on engineered bacteria by inhibiting mazE expression at 28℃ did not reach our expectation. We obtained two hypotheses by analyzing the experimental results and literature.

1. The concentration gap between MazE and MazF caused by RBS inhibition of MazE at 28℃ was not enough to kill the engineered bacteria quickly. Moreover, the engineered bacteria had negative feedback regulation of MazEF in their chromosomes, and the up-regulation of maze expression by the negative feedback regulation mechanism made the engineered bacteria free from MazF killing after several hours of culture.

2. According to some literatures, the effect of MazF is growth arrest rather than programmed cell death. Therefore, it is possible that in the case of dormancy of engineered bacteria, we placed them at 37 ℃ after coating, mazE expression was restored, and engineered bacteria were released from growth inhibition.

We wanted to verify the second hypothesis, so the engineered bacteria were over-diluted and coated on LB-Kanr solid medium and cultured at 28 ℃, and then a petri dish was taken every hour and placed at 37 ℃ for a total of 8 hours to see if single colony can recover at better circumstances. Colonies were then incubated for 5 hours to be seen. As the outcome shows, all two colonies treated for less than 7 hours recovered compared to the control group grown in 28℃. And those treated for more than 6 hours suffer from proliferation inhibition to different degrees. However, low concentration leads to high possibility of coincidences, further experiments of higher concentration will be conducted later and results of our current exploration are shown below in Fig. 22.

![](https://static.igem.wiki/teams/4117/wiki/images/result/new.png)

Fig.22 Comparison of growth of the engineered bacteria for different time cultured at 28℃( (A) 5h (B) 6h (C) 7h (D) the control group) and then recovered at 37℃, the initial dilution of the bacterial solution is the same .

##### RNA Thermometer Verification Pathway

To verify the function of RNA thermometers, strains were cultured at 37 °C and 28 °C respectively, and the initial OD600 and GFP fluorescence intensity were measured. Then samples were taken every half an hour to measure the OD600 and GFP fluorescence intensity.

GFP measurement results are shown in Fig.23. And we used a fluorescence microscope for observation. The bacterial liquid under the fluorescence microscope is as Figure 24:

![](https://static.igem.wiki/teams/4117/wiki/images/result/23.png)

Fig.23 GFP fluorescence intensity after incubating in different temperatures.

![](https://static.igem.wiki/teams/4117/wiki/images/result/24.png)

Fig.24 Images under fluorescene microscope. (A) No GFP in engineerd bacteria incubated at 28℃. (B) Bright field image of bacteria incubated at 28℃. (C) The field of view after superposition of A and B. (D) GFP in engineerd bacteria incubated at 37℃. (E) Bright field image of bacteria incubated at 37℃. (F) The field of view after superposition of D and E. (fluorescene microscope 10 x 100).

![](https://static.igem.wiki/teams/4117/wiki/images/toppic.png)

{% include 'pages/new\_footer.html' %}