iGEM 2021 — Team BUCT

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# Team:BUCT/Notebook

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* [June](#June)
* [July](#July)
* [August](#August)
* [September](#September)

Notebook

June

**6.11** We read the literature on suicide system and realized that MazEF is a pair of very valuable TA systems that can be applied to our project.

**6.17** We thought about how to select "variables" as the switch of the suicide system, and finally selected "changes in oxygen concentration", and found a suitable oxygen promoter-phyb on NCBI.

July

**7.10** We checked the literature to find the source of the target gene.

**7.16** We obtained the MazF from the HZAU-China team as the toxin gene.

**7.19** We got the endogenous E.coli MazE and MazF from E.coli BW25113 through PCR.

**7.21** We designed primers and started the construction of suicide plasmids.

August

**8.11** We built 5-HTP and suicide plasmids, and got through PCR, Agarose gel electrophoresis , gel recycle, enzyme digestion, ligation, and chemical transfer for PCD, mazE/F, trpE/C/B, and P4H.

**8.12** We verified the plasmid and found that there was a problem with the pSA plasmid, and carried out test-tube culture of bacteria containing pSA.

**8.13** The lac-mazF-T1-phyb-mazE-T1 suicide plasmid was successfully constructed, and was successfully verified.

**8.14** We extracted the plasmid of the pCS-lac-mazF bacterial solution and verified that it was successful. Half of the suicide parts have been constructed correctly. We also inoculated pSA empty plasmid and pSA-lac-TrpB-P4H-PCD bacterial solution.

**8.15** We extracted the pSA-lac-TrpB-P4H-PCD plasmid, verified by restriction enzyme digestion, and used pSA to construct a three-gene plasmid.

**8.16** We extracted the vgb (plasmid)+mazE, pSA+P4H+PCD+TrpB plasmid, verified by restriction enzyme digestion.

**8.17** We got the suicide plasmid sequencing result, which is correct.

**8.18** We successfully constructed the vgb-mazE plasmid and ligated it with lac-mazF.

**8.20** We carry out PCD amplification on PCD, P4H, and TrpB, and carry out gel recycle.

**8.21** The extracted plasmid was successfully verified, and homologous recombination bacteria were cultivated.

**8.22** We used the homologous recombination method pCS-lac-TrpB-P4H-PCD to extract the plasmid, and the restriction enzyme digestion verification was successful.

**8.23** We successfully prepared high-efficiency Nissle 1917 competent, homologous recombination of lac-FadD-FadL plasmid and pCS-lac-gadB(mut)-gdhA plasmid, and connected pSA-AnTrpC-TrpE and lac-TrpB-P4H-PCD.

**8.24** We introduced the plasmid into the target bacteria Nissle 1917.

**8.25** We cultivated engineering bacteria.

**8.26** Extract the plasmid.

**8.27** We performed PCR again on the lac-TrpB-P4H-PCD-T1 frame and saved the strain.

**8.28** We used the plasmid extracted from the bacteria as a template, and used FadD primers to verify the successful construction of the plasmid.

**8.29** We combined 5-HTP plasmid (pSA-AnTrpC-TrpE-lac-TrpB-P4H-PCD) and also combined GABA & FadD-Fad (pCS-lac-gadB(mut)-gdhA-lacFadD-FadL)

**8.30** We revisef the suicide part verification plan, verifying that "death can be induced under aerobic conditions" failed, and prepared to rebuild.

September

**9.1** We learnt how to use anaerobic workstation to create an anaerobic environment for the growth of engineering bacteria, which was beneficial to the characterization of suicide system.

**9.2** We homologous recombined pSA-lac-TrpEG-AnTrpC and lac-TrpB-P4H-PCD.

**9.3** We reconstructed 5-HTP plasmid construction-homologous recombination system.

**9.5**We used two methods to construct the general flow chart of 5HTP framed plasmid simultaneously.

**9.7**We constructed suicide parts plasmid Gibson.

**9.8** We carried out the transformation and coating of plasmid.

**9.9** We activated Nissle bacteria, and constructed 5HTP framed plasmid by restriction enzyme ligation.

**9.10** We prepared Nissle bacterial liquid, and made GABA fermentation to prepare pCS-gadB(mut)-gdhA.

**9.14** We cultured nissle 1917 that was successfully introduced into plasmid in glove box.

**9.15** We verified PCs-gadb (mut)-gdha-lac-fadd-fadl, the electrotransformed double plasmid 5HTP.

**9.16** We constructed GABA+fad- plasmid.

**9.17** We verified the previous plasmid by enzyme digestion, but the verification failed, and 5-HTP was ready to electrotransfer double plasmids.

**9.18** We constructed 5HTP fermentation-double plasmid, which was no longer closed, but directly double-transformed, and it produced 5HTP, which was successfully verified by enzyme digestion.

**9.19** We successfully constructed GABA+fad- plasmid.

**9.20** We introduced 5HTP fermentation-double plasmid to prepare competent cells.

**9.21** We successfully carried out GABA+fad fermentation-electrotransfer coating.

**9.22** We carried out 5-HTP fermentation.

**9.23** We used colorimetry to detect GABA fermentation, and the result was not satisfactory.

**9.24** We tested GABA fermentation.

**9.25** After constantly exploring the reaction conditions, we found that Nissle 1917 needs to grow very slowly in anaerobic environment, and it takes 48-72 hours to see obvious colonies, thus confirming the success of suicide system.

*The whole **BUCT** team would like to thank our sponsors. Especially:*

[![](https://static.igem.org/mediawiki/2021/9/9c/T--BUCT--foot<p><b>9.jp</b>g)](https://www.snapgene.com/)
[![](https://static.igem.org/mediawiki/2021/a/af/T--BUCT--foot5.jpg)](https://www.buct.edu.cn/)
[![](https://static.igem.org/mediawiki/2021/e/e7/T--BUCT--foot2.jpg)](https://www.fmtbio.com/)
[![](https://static.igem.org/mediawiki/2021/7/7d/T--BUCT--foot6.jpg)](http://www.yakult.com.cn/)
[![](https://static.igem.org/mediawiki/2021/e/e3/T--BUCT--foot7.jpg)](http://www.cau.edu.cn/)
[![](https://static.igem.org/mediawiki/2021/f/f6/T--BUCT--BUCTfoot10.png)](https://ww2.mathworks.cn/)

*Tel: 86+18718806286*

*Address: No.15 North Third Ring East Road,Chaoyang District,Beijing,P.R.China. **Beijing University of Chemical Technology***

![](https://static.igem.org/mediawiki/2021/7/7a/T--BUCT--BUCTfoot11.png) ***BUCT--iGEM***

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# Team:BUCT/Engineering

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* [Overview](#Overview)
* [1. 5HTP synthesis](#1. 5HTP synthesis)
* [2. Enhances beta
  oxidation](#2. Enhances beta oxidation)
* [3. Oxygen-regulated
  suicide](#3. Oxygen-regulated suicide)
* [References](#References)

Overview

![](https://static.igem.org/mediawiki/2021/d/dd/T--BUCT--overview.png)

"We followed the engineering cycle for countless iterations over the course of our project.

1. 5HTP synthesis

![](https://static.igem.org/mediawiki/2021/1/1a/T--BUCT--5-HTP.png)

2. Enhances beta oxidation

![](https://static.igem.org/mediawiki/2021/7/79/T--BUCT--Enhance.png)

3. Oxygen-regulated suicide

![](https://static.igem.org/mediawiki/2021/0/02/T--BUCT--oxygen.png)

## [Please review the Result page for more information](https://2021.igem.org/Team%3ABUCT/Results)

References

***1. Lin Y, Sun X, Yuan Q, Yan Y. Engineering bacterial phenylalanine 4-hydroxylase for microbial synthesis of human neurotransmitter precursor 5-hydroxytryptophan. ACS Synth Biol. 2014 Jul 18;3(7):497-505. doi: 10.1021/sb5002505. Epub 2014 Jun 30. PMID: 24936877.
2. Wang H, Liu W, Shi F, Huang L, Lian J, Qu L, Cai J, Xu Z. Metabolic pathway engineering for high-level production of 5-hydroxytryptophan in Escherichia coli. Metab Eng. 2018 Jul;48:279-287. doi: 10.1016/j.ymben.2018.06.007. Epub 2018 Jun 19. PMID: 29933064.
3. Luo H, Schneider K, Christensen U, Lei Y, Herrgard M, Palsson BØ. Microbial Synthesis of Human-Hormone Melatonin at Gram Scales. ACS Synth Biol. 2020 Jun 19;9(6):1240-1245. doi: 10.1021/acssynbio.0c00065. Epub 2020 Jun 5. PMID: 32501000.
4. Chae TU, Ko YS, Hwang KS, Lee SY. Metabolic engineering of Escherichia coli for the production of four-, five- and six-carbon lactams. Metab Eng. 2017 May;41:82-91. doi: 10.1016/j.ymben.2017.04.001. Epub 2017 Apr 5. PMID: 28390749.
5. Wu H, Wang H, Chen J, Chen GQ. Effects of cascaded vgb promoters on poly(hydroxybutyrate) (PHB) synthesis by recombinant Escherichia coli grown micro-aerobically. Appl Microbiol Biotechnol. 2014 Dec;98(24):10013-21. doi: 10.1007/s00253-014-6059-y. Epub 2014 Sep 13. PMID: 25216582.
6. J. Agric. Food Chem. 2017, 65, 8, 1669–1676Publication Date:February 2, 2017<https://doi.org/10.1021/acs.jafc.6b05237>
7. Nikolic, N., Bergmiller, T., Vandervelde, A., Albanese, T. G., Gelens, L., & Moll, I. (2018). Autoregulation of mazEF expression underlies growth heterogeneity in bacterial populations. Nucleic acids research, 46(6), 2918–2931. <https://doi.org/10.1093/nar/gky079>
8. Engelberg-Kulka, H., Hazan, R., & Amitai, S. (2005). mazEF: a chromosomal toxin-antitoxin module that triggers programmed cell death in bacteria. Journal of cell science, 118(Pt 19), 4327–4332. <https://doi.org/10.1242/jcs.02619>
9. Hazan R, Sat B, Engelberg-Kulka H. Escherichia coli mazEF-mediated cell death is triggered by various stressful conditions. Journal of Bacteriology. 2004 Jun;186(11):3663-3669. DOI: 10.1128/jb.186.11.3663-3669.2004. PMID: 15150257; PMCID: PMC415763.***

*The whole **BUCT** team would like to thank our sponsors. Especially:*

[![](https://static.igem.org/mediawiki/2021/9/9c/T--BUCT--foot9.jpg)](https://www.snapgene.com/)
[![](https://static.igem.org/mediawiki/2021/a/af/T--BUCT--foot5.jpg)](https://www.buct.edu.cn/)
[![](https://static.igem.org/mediawiki/2021/e/e7/T--BUCT--foot2.jpg)](https://www.fmtbio.com/)
[![](https://static.igem.org/mediawiki/2021/7/7d/T--BUCT--foot6.jpg)](http://www.yakult.com.cn/)
[![](https://static.igem.org/mediawiki/2021/e/e3/T--BUCT--foot7.jpg)](http://www.cau.edu.cn/)
[![](https://static.igem.org/mediawiki/2021/f/f6/T--BUCT--BUCTfoot10.png)](https://ww2.mathworks.cn/)

*Tel: 86+18718806286*

*Address: No.15 North Third Ring East Road,Chaoyang District,Beijing,P.R.China. **Beijing University of Chemical Technology***

![](https://static.igem.org/mediawiki/2021/7/7a/T--BUCT--BUCTfoot11.png) ***BUCT--iGEM***

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# Team:BUCT/Experiments

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* [Protocol](#Protocol)

Experiments

Protocol

This year the team BUCT has done a lot of experiments. From constructing plasmid to ferment, a whole bunch of lab operations and lab skills involved in. Below is our protocol of experiment of this year:

*The whole **BUCT** team would like to thank our sponsors. Especially:*

[![](https://static.igem.org/mediawiki/2021/9/9c/T--BUCT--foot9.jpg)](https://www.snapgene.com/)
[![](https://static.igem.org/mediawiki/2021/a/af/T--BUCT--foot5.jpg)](https://www.buct.edu.cn/)
[![](https://static.igem.org/mediawiki/2021/e/e7/T--BUCT--foot2.jpg)](https://www.fmtbio.com/)
[![](https://static.igem.org/mediawiki/2021/7/7d/T--BUCT--foot6.jpg)](http://www.yakult.com.cn/)
[![](https://static.igem.org/mediawiki/2021/e/e3/T--BUCT--foot7.jpg)](http://www.cau.edu.cn/)
[![](https://static.igem.org/mediawiki/2021/f/f6/T--BUCT--BUCTfoot10.png)](https://ww2.mathworks.cn/)

*Tel: 86+18718806286*

*Address: No.15 North Third Ring East Road,Chaoyang District,Beijing,P.R.China. **Beijing University of Chemical Technology***

![](https://static.igem.org/mediawiki/2021/7/7a/T--BUCT--BUCTfoot11.png) ***BUCT--iGEM***

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![](https://static.igem.org/mediawiki/2021/e/ed/T--BUCT--BUCTQRcode.jpg)

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# Team:BUCT/Results

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* [Excellence](https://2021.igem.org/Team%3ABUCT/Excellence_in_Another_Area)

* [PART 1](#PART 1)
* [PART 2](#PART 2)
* [PART 2.1](#PART 2.1)
* [PART 2.2](#PART 2.2)
* [PART 3](#PART 3)
* [PART 4](#PART 4)

PART 1: Fermentation result of GABA

Below are some pictures showing that the process of fermenting GABA.

![](https://static.igem.org/mediawiki/parts/6/60/T—BUCT—GABA-甘-1.jpg)
![](https://static.igem.org/mediawiki/parts/b/b6/T—BUCT—GABA-甘-2.jpg)
![](https://static.igem.org/mediawiki/parts/7/71/T—BUCT—GABA-甘-3.jpg)
![](https://static.igem.org/mediawiki/parts/0/04/T—BUCT—GABA-甘-4.jpg)

After fermentation, we detect the data by using Chromatograph software.

Below are some detail information about the machine and methods we used.
MACHINE: Chromatograph software name:LCsolution 15C
                  MODEL:SPD-15C
                  CAT.No:465-04197-91
                  SERIAL No:L20525031877CS
                  VOL TS:220V-240～50-60Hz 150VA

**Method:**

Before measuring **GABA** output, we first carried out the derivative steps:

1. Take standard sample solution and diluted sample and put 200ul in 1.5ml test tube

2. Add 100ul 1M triethylamine-acetonitrile solution and 100ul 0.1m phenyl isothiocyanate-acetonitrile solution successively, mix them evenly, and stand for 1h at room temperature.

3. Add 200ul n-hexane and mix it evenly. After vortex mixing, stand for 30min at room temperature, and stand for stratification

4. Use a syringe to absorb the derivativated lower liquid, and put it into liquid and vial after passing 0.22um organic phase filtration membrane.

 **Below is the result of fermentation and it shows that we have successfully produced GABA from simple carbon sources.**

**GABA was produced by fermentation with glycerol as substrate:**

![](https://static.igem.org/mediawiki/parts/a/a7/T--BUCT--GABA_was_produced_by_fermentation_with_glycerol_as_substrate.png)

**GABA was produced by fermentation with palm oil as substrate:**

![](https://static.igem.org/mediawiki/parts/8/84/T--BUCT--GABA_was_produced_by_fermentation_with_plam_oil_as_substrate.png)

****GABA was produced by fermentation with soybean oil as substrate”****

![](https://static.igem.org/mediawiki/parts/4/4f/T--BUCT--GABA_was_produced_by_fermentation_with_soybean_oil_as_substrate.png)

**Analysis:**

As we can see from the three charts, we overproduce GABA up to **5.276g/L** when the substrate is **glycerol**. And overproduce GABA up to **4.807g/L** when the substrate is **palm oil** and up to **8.315g/L** when the substrate is **soybean oil**.

Based on these statistics, it is obvious that we have successfully **produce GABA**. Also, considering that normal synthesis pathways, which consisted of ***gadB*(wild)**, of producing GABA usually produce GABA for **1mg/ml**, so it is obvious that our *gadB*(mut) gene plays its special function and helps overproducing GABA!

PART 2: Fermentation result of
*pCS-gadB(mut)-gdhA-fadD-fadL-T1* **and** *pCS-gadB(mut)-gdhA-fadD-fadL-T1(∆fadR)*

We constructed the plasmid *pCS-gadB(mut)-gdhA-fadD-fadL-T1* and introduced it into *E.coli*. After fermentation, we detected the consumption of palm oil and the production of GABA.

PART 2.1: Detect the consumption of palm oil

We configure the Gas Chromatograph test group: 200µl fermentation broth (obtain 20g/l palm oil), 2000µl petroleum ether-diethyl ether, 100µl methanol-KOH. From the chart, we can see the amount of palm oil is decreasing, and it proves this composite part **lac-fadD-fadL-T1** ( [BBa\_K3875001](http://parts.igem.org/Part%3ABBa_K3875001)) (Wild Strain) can enhance the decomposition of fatty acids by beta oxidation in *E.coli*.

Then we cut fadR which represses the expression of above 15 genes, constructing ∆fadR+lac-fadD-fadL-T1(∆fadR Strain). We find the amount of palm oil decreasing more quickly, and the rate of decline literally improved.

![](https://static.igem.org/mediawiki/parts/a/af/T—BUCT—fermantation_time.jpg)
![](https://static.igem.org/mediawiki/parts/8/84/T—BUCT—fermantation_time-2.jpg)
![](https://static.igem.org/mediawiki/parts/4/45/T—BUCT—strain.jpg)

We can notice that the ∆fadR strain shows a weaker culture performance than the wild strain in culture dishes. The value of OD600 of ∆fadR strain less than wild strain as well. However, in the beginning 24h, the ∆fadR strain consumes more palm oil than wild strain. That is a good explanation that our new construction is successful. Linking fadL and fadD and cutting fadR can literally enhance the decomposition of fatty acids by beta oxidation in *E.coli*.

![](https://static.igem.org/mediawiki/parts/4/4d/T—BUCT—the_states_of_samples_60h.jpg)
![](https://static.igem.org/mediawiki/parts/9/98/T—BUCT—the_states_of_samples_72h.jpg)

PART 2.2: Detect the consumption of palm oil.

Production of GABA with *pCS-gadB(mut)-gdhA-fadD-fadL-T1*:

![](https://static.igem.org/mediawiki/parts/8/87/T—BUCT—fermantation_time-3.jpg)

Production of GABA with *pCS-gadB(mut)-gdhA-fadD-fadL-T1 (∆fadR)*:

![](https://static.igem.org/mediawiki/parts/0/0b/T—BUCT—fermantation_time-4.jpg)

As we can see from these two charts, we got a good production of GABA. **However, the bacteria could not continue to produce GABA due to insufficient substrates and GABA's self-decomposition, so the production of GABA decreased after 48 hours**.

**Based on Part 2, it is obvious that we have successfully produces GABA while consuming fat**.

PART 3: Production of 5-HTP

**Result-5-HTP**

We coated *E. coli* with plasmids pCS-lac-trpB-p4h-pcd-T1 and pSA-lac-trpEG-AntrpC, and the results were as follows:

![](https://static.igem.org/mediawiki/parts/f/f4/T—BUCT—psa.jpg)

It is possible that the synthesis path of 5-HTP is too long, so the yield of 5-HTP is not high.

The fermentation production of 5-HTP in E. coli Nissle 1917 appeared rising trend, and the yield at 48h was about 2mg/L. Technically, blocking chorismate's entry into the branching pathway for synthesis of other substances could further increase the yield of 5-HTP. It’s also a question we needs to solve in the future.

![](https://static.igem.org/mediawiki/parts/8/8a/T—BUCT—fermantation_time-5.jpg)

***Figure. 1** 5-HTP fermentation curve with glycerol as substrate*

PART 4: Toxin-antitoxin system

Nissle 1917, which was successfully imported into the plasmids, was cultured in the anaerobic workstation.

![](https://static.igem.org/mediawiki/parts/5/58/T—BUCT—teemperature.jpg)

***Figure.**  The engineered bacteria are cultured under oxygen-free conditions*

Cultured 16h under aerobic conditions, no colonies were found to grow, which proved that there was no escape of engineering bacteria under aerobic conditions, which is in good compliance with the safety principle.

![](https://static.igem.org/mediawiki/parts/9/94/T—BUCT—oxggen-16.jpg)

***Figure.**  Aerobic conditions cultured 16h, engineering bacteria died*

After constantly groping for reaction conditions, it was found that *E coli.* Nissle 1917 grew very slowly in an oxygen-free environment, requiring 48-72h growth in order to see the obvious colony of the crucible in the aerobic air environment, it is not possible to observe obvious traces of engineering bacteria growth, which can indicate the success of the suicide system.

![](https://static.igem.org/mediawiki/parts/4/41/T—BUCT—oxggen-48.jpg)

***Figure.**  The culture contrast between aerobic environment and anaerobic environmentd*

*The whole **BUCT** team would like to thank our sponsors. Especially:*

[![](https://static.igem.org/mediawiki/2021/9/9c/T--BUCT--foot9.jpg)](https://www.snapgene.com/)
[![](https://static.igem.org/mediawiki/2021/a/af/T--BUCT--foot5.jpg)](https://www.buct.edu.cn/)
[![](https://static.igem.org/mediawiki/2021/e/e7/T--BUCT--foot2.jpg)](https://www.fmtbio.com/)
[![](https://static.igem.org/mediawiki/2021/7/7d/T--BUCT--foot6.jpg)](http://www.yakult.com.cn/)
[![](https://static.igem.org/mediawiki/2021/e/e3/T--BUCT--foot7.jpg)](http://www.cau.edu.cn/)
[![](https://static.igem.org/mediawiki/2021/f/f6/T--BUCT--BUCTfoot10.png)](https://ww2.mathworks.cn/)

*Tel: 86+18718806286*

*Address: No.15 North Third Ring East Road,Chaoyang District,Beijing,P.R.China. **Beijing University of Chemical Technology***

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