iGEM 2022 — Team buct

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## What should this page contain?

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* 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

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* [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)

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{% block title %}ENGINEERING{% endblock %}
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[Overview](#section1)

[1 Synthesis of 2-phenylethyl alcohol (2-PE)](#section2)

[2 Synthesis of Caffeine](#section3)

[3 Suicide system](#section4)

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# Overview

In the process of engineering practice, we are constantly thinking about how to improve the synthesis of 2-PE and caffeine to make the project more consistent with real-world prototypes. We also think very carefully about biosafety. In each iteration and update, we are very grateful to the people who provide us with help and advice.

# 1 Synthesis of 2-phenylethyl alcohol (2-PE)

| Stage 1 | Stage 2 | Stage 3 |
| --- | --- | --- |
| In the early stage of 2-PE synthesis and construction, we did not go well.  Our 2-PE synthesis involves three genes: aroG, adh6 and aro10. In the process of constructing three genes on the same plasmid, we found that pET could not lay down all three genes at the same time.  It can be speculated that the length of three genes placed on a plasmid is close to the length that the plasmid can bear, which may be because the molecular weight of the plasmid is too large, resulting in low transformation efficiency. | The construction of aroG, adh6 and aro10 genes on plasmid pET failed, so we temporarily used plasmid pCS to express aroG and plasmid pET to express adh6 and aro10. After the successful construction of pET-aro10-adh6 and pCS-aroG, 2-PE was synthesized by E. coli Nissle 1917 through induced expression and fermentation.  After 72h fermentation, the content of 2-PE was detected. | The highest yield we obtained was 67.16mg/L. Compared with the maximum yield of JLU-China, the yield of BUCT was lower. Referring to the ideal yield of 130mg/L calculated by JLU-China, we believe that measures should be taken to increase the yield of 2-PE.  First, add phenylalanine (Phe) to the substrate; Second, the genes applied by JLU-China in 2-PE synthesis were compared with our gene combination to select the optimal element combination. |
| --- | --- | --- |

Unfortunately, due to the epidemic, we could not use our laboratory for a long time. In the future, we will continue to complete the new design.

# 2 Synthesis of Caffeine

| Stage 1 | Stage 2 |
| --- | --- |
| We constructed pCS-TcAncCS-TcCS2-TCS1 based on methylated proteases selected from the literature, starting with Xanthine for methylation, and then using 3-methylxanthine to synthesize theobromine, a precursor of caffeine, and finally caffeine.  After induction and fermentation, caffeine content was measured by HPLC. The maximum yield of theobromine and caffeine was 7.16mg/L and 1.83mg/L, respectively. | In reality, there was no condition to verify the yield of E. coli Nissle in practical application environment. We need to test the reasonableness of the yield through modeling and calculation.  According to the results of the model, the caffeine production in the actual application environment can exceed the effective concentration threshold of 0.4mg/L. So in reality, our engineered bacteria can produce caffeine to promote the growth of hair follicles. |
| --- | --- |

# 3 Suicide system

| Stage 1 | Stage 2 | Stage 3 |
| --- | --- | --- |
| The original suicide system requires iron concentration to control promoter expression. And the promoter controls the expression of the toxic protein MazF. We constructed this initial model in the hope of finding an economical, safe and effective donor of iron ions. We also consulted two doctors and took other teams' advice.  Finally, we chosed to try the quorum sensing promoter because the safety of this prototype was not high enough. | We combined several suggestions and then built a quorum-sensing promoter-controlled suicide system. This promoter also controls the expression of MazF.  In order to screen a reasonable bacterial density, we calculated on the basis of laboratory fermentation data.  We then determined whether caffeine could be synthesized at the desired yield under real-world conditions to ensure that the bacterial density selection did not fall below that standard. | We considered the competitive relationship between E. coli Nissle 1917 and Malassezia grown under the same conditions. In our design, Nissle is required to inhibit Malassezia by competing for free fatty acids.  According to calculation, when triggering OD=0.21, MazF would be expressed and cause Nissle to die.  Meanwhile, under 0.21, the growth curve of Nissle is always higher than that of Malassezia, indicating that Nissle could inhibit Malassezia. The modeling and calculation based on the experimental data proved that our Nissle could not only ensure the beneficial effect of caffeine on hair follicles at 250CFU/mm2, but also compete with Malassezia for survival resources at this density and affect the growth of Malassezia. |
| --- | --- | --- |

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[Synthesis of Caffeine and 2-Phenylethyl Alcohol](#section1)

[Increased fatty acid metabolism](#section2)

[Reference](#section3)

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## Synthesis of Caffeine and 2-Phenylethyl Alcohol

The overall experimental process from objective gene to genetically engineered bacteria:

Gene purchase → plasmid construction → competent transformation → plasmid extraction → agarose gel electrophoresis → gel recovery → enzyme digestion → preparation of E.coli Nissle 1917 competence→ competent transformation → culture and fermentation → yield detection.

1. Purchase genes

The essential genes, TCS1, TcAncCS2 and TcCS2 for caffeine and aroGfbr, aro10, adh6 for 2-PE were purchased from biotech companies.

2. Construct plasmids

Plasmids were constructed by enzymatic digestion and ligation.

![](https://static.igem.wiki/teams/4146/wiki/wiki-test/experiment/fig1.png)

pET-aro10-adh6

![](https://static.igem.wiki/teams/4146/wiki/wiki-test/experiment/fig2.png)

pCS-aroGfbr

![](https://static.igem.wiki/teams/4146/wiki/wiki-test/experiment/fig3.png)

pCS-TCS1

3. Competency transformation: Commercial E. coli DH5a was used for competent transformation and plasmid amplification.

4. Extract plasmid.

5. Enzyme validation

6. Agarose gel electrophoresis

![](https://static.igem.wiki/teams/4146/wiki/wiki-test/experiment/fig4.png)

Figure 1 Validation of caffeine plasmid (All are correct)

![](https://static.igem.wiki/teams/4146/wiki/wiki-test/experiment/fig5.png)

Figure 2 Validation of 2-PE plasmid (L7-L9 are correct)

7. Glue recovery

Note: When constructing the plasmids for 2-PE , we tried to add three genes (aro10, adh6 and aroG) on the high-copies pZE plasmid , but the construction process proved unsuccessful , probably due to the length of the plasmid.

8. Enzyme digestion

9. Preparation of E.coli Nissle 1917 COMPETENCE

10. Electroporate, and cultivate in LB

![](https://static.igem.wiki/teams/4146/wiki/wiki-test/experiment/fig6.jpg)

Figure 3 Strains for 2-PE

![](https://static.igem.wiki/teams/4146/wiki/wiki-test/experiment/fig7.jpg)

Figure 4 Strains for Caffeine

11. Culture and fermentation

[M9 medium formula: 1g/L MOPS, 2.5g/L yeast powder, 5.64g/L M9 Minimal Salt, 10g/L glucose, 1ml MgSO4·7H2O (1 M), and 50mL calcium chloride (1 M)]

Start fermentation in shake flasks. Add inducer IPTG when the OD600≈0.6-0.8, sample every 12 hours to determine OD value, and keep it for HPLC. Incubate at 37℃ for 2h, add IPTG for induction, and transfer it to a shaking table at 30℃.

12 hours to determine OD value, and keep it for HPLC. Incubate at 37℃ for 2h, add IPTG for induction, and transfer it to a shaking table at 30℃.

13. Test out (HPLC):

2-PE: 1 ‰ trifluoroacetic acid as mobile phase, acetonitrile as organic phase

CA: 2 ‰ formic acid as mobile phase, acetonitrile as organic phase

## Increased fatty acid metabolism

Knock out competence gene by using CRISPR/Cas9 system's ability to edit and regulate DNA in various organisms and cell types.

| Reagents: | Equipment: |
| --- | --- |
| . E.coli nissle 1917  . Plasmid pTarget\_ competence\_ N20  . Strain culture liquid | . PCR tubes  . Electrophoresis apparatus  . PCR instrument  . 37 ℃ incubator |
| --- | --- |

1、As shown in the figure, first is the schematic diagram of the principle that we use CRISPR to knock out the gene competence. Our project hopes to enhance the β- Oxidation, so we reduced the competence that inhibited the expression of the above 15 genes. N20 sequence is 5 '- GGAATACCGCTCCC-3'. This sequence will help Cas9 protein and gRNA complex locate knockout sites. When Cas9 protein finds the target sequence of PAM, it will immediately melt the base upstream of PAM and pair it with the complementary region on the guide RNA. If the complementary region and target region are correctly paired, RuvC and HNH nuclease domains will cleave target DNA after the third nucleotide base upstream of PAM.

![](https://static.igem.wiki/teams/4146/wiki/wiki-test/experiment/fig8.png)

2、We need to design two plasmids. One is pCas9, which produces Cas9 protein, and the other is pTarget, which transcribes sgRNA. Then guide it into E.coli Nissle 1917. We introduced the design plasmid, and then mixed the bacteria for shaking table culture, and kept them in the 37 ℃ incubator for 24 hours.

3、PCR experiment was used to detect the new N20 sequence in the Ptarget plasmid. The results showed that we successfully imported the N20 sequence - 5 '- GGATA ACCGCTCCCC-3'.

![](https://static.igem.wiki/teams/4146/wiki/wiki-test/experiment/fig9.png)

Figure 5 PCR the new N20 sequence in Ptarget plasmid

4、When Cas9 protein finds the target sequence of PAM, it will immediately melt the base upstream of PAM and pair it with the complementary region on the guide RNA. If the complementary region and target region are correctly paired, RuvC and HNH nuclease domains will cleave target DNA after the third nucleotide base upstream of PAM. At this time, the PCR comparison analysis is performed again to determine whether the gene knockout is successful.

![](https://static.igem.wiki/teams/4146/wiki/wiki-test/experiment/fig10.png)

Figure 6 Knocking out of competence

5、The following figure is our design introduction plasmid diagram.

![](https://static.igem.wiki/teams/4146/wiki/wiki-test/experiment/fig11.png)

Figure 7 Plasmid of pTarget

BUCT basic experimental operation process:

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## Reference

Ashihara H, Sano H, Crozier A. Caffeine and related purine alkaloids: biosynthesis, catabolism, function and genetic engineering. Phytochemistry. 2008 Feb;69(4):841-56. doi: 10.1016/j.phytochem.2007.10.029. Epub 2007 Dec 19. PMID: 18068204.

Motamayor JC, Mockaitis K, Schmutz J, Haiminen N, Livingstone D 3rd, Cornejo O, Findley SD, Zheng P, Utro F, Royaert S, Saski C, Jenkins J, Podicheti R, Zhao M, Scheffler BE, Stack JC, Feltus FA, Mustiga GM, Amores F, Phillips W, Marelli JP, May GD, Shapiro H, Ma J, Bustamante CD, Schnell RJ, Main D, Gilbert D, Parida L, Kuhn DN. The genome sequence of the most widely cultivated cacao type and its use to identify candidate genes regulating pod color. Genome Biol. 2013 Jun 3;14(6):r53. doi: 10.1186/gb-2013-14-6-r53. PMID: 23731509; PMCID: PMC4053823.

Jin JQ, Yao MZ, Ma CL, Ma JQ, Chen L. Natural allelic variations of TCS1 play a crucial role in caffeine biosynthesis of tea plant and its related species. Plant Physiol Biochem. 2016 Mar;100:18-26. doi: 10.1016/j.plaphy.2015.12.020. Epub 2016 Jan 4. PMID: 26773541.

Balachandran N, Heimhalt M, Liuni P, To F, Wilson DJ, Junop MS, Berti PJ. Potent Inhibition of 3-Deoxy-d-arabinoheptulosonate-7-phosphate (DAHP) Synthase by DAHP Oxime, a Phosphate Group Mimic. Biochemistry. 2016 Dec 6;55(48):6617-6629. doi: 10.1021/acs.biochem.6b00930. Epub 2016 Nov 17. PMID: 27933795.

Goffeau A, Barrell BG, Bussey H, Davis RW, Dujon B, Feldmann H, Galibert F, Hoheisel JD, Jacq C, Johnston M, Louis EJ, Mewes HW, Murakami Y, Philippsen P, Tettelin H, Oliver SG. Life with 6000 genes. Science. 1996 Oct 25;274(5287):546, 563-7. doi: 10.1126/science.274.5287.546. PMID: 8849441.

Goffeau A, Barrell BG, Bussey H, Davis RW, Dujon B, Feldmann H, Galibert F, Hoheisel JD, Jacq C, Johnston M, Louis EJ, Mewes HW, Murakami Y, Philippsen P, Tettelin H, Oliver SG. Life with 6000 genes. Science. 1996 Oct 25;274(5287):546, 563-7. doi: 10.1126/science.274.5287.546. PMID: 8849441.

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[Overview](#section1)

[Fatty acid consumption](#section2)

[Synthesis of 2-Phenylethyl Alcohol (2-PE)](#section3)

[Synthesis of Caffeine](#section4)

[Yield prediction and promoter screening](#section5)

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# Overview

BUCT mainly requires the synthesis of caffeine and 2-phenylethyl alcohol (2-PE) on the basis of fatty acid consumption. We successfully knocked out a gene that inhibits fatty acid oxidation in E. coli, thereby enhancing the free fatty acid consumption function of E. coli Nissle 1917. After the first-generation synthesis of 2-PE, we compared the yield with JLU-China and set the second-generation plan. The synthetic yield of caffeine is low, but we do see some accumulation of the precursor theobromine.

Based on the comprehensive consideration of the growth and effective yield of E. coli Nissle 1917, we also calculated and modeled the highest bacterial density that could inhibit Malassezia and satisfy the effective amount of product.

# Fatty acid consumption

We have successfully knocked out the fadR in E. coli Nissle 1917 through CRISPR/Cas9.

The strain was also used for the synthesis of 2-phenylethyl alcohol and caffeine. According to the growth monitoring data of this strain in the fermentation process, we fitted a growth curve to calculate the yield of E. coli Nissle 1917 in the actual process.

# Synthesis of 2-Phenylethyl Alcohol (2-PE)

![](https://static.igem.wiki/teams/4146/wiki/wiki-test/result/fig1.jpg)

Figure 1. 2-PE fermentation samples

The first round of 2-PE synthesis has been completed. The concentration of the product can be calculated by HPLC. E. coli Nissle 1917 was able to synthesize 2-PE successfully by adding glucose, which indicated that we successfully opened the synthesis path in Nissle, although the yield of BUCT in de novo synthesis was only about 60% of that in JLU-China, which also synthesized 2-PE. However, the small molecule synthesis of aromatic phenols, which we used as an example of 2-PE, can be used as a prerequisite for more flavor modifications in the future.

![](https://static.igem.wiki/teams/4146/wiki/wiki-test/result/fig2.jpg)

Figure 2. Standard curve for measurement of 2-PE by HPLC

The confidence of the 2-PE standard curve is very high, and we can directly filter the samples after fermentation without derivatization.

![](https://static.igem.wiki/teams/4146/wiki/wiki-test/result/fig3.jpg)

Figure 3. Yield of 2-PE fermentation in E. coli Nissle 1917

In the future, the synthesis of 2-PE will continue to be optimized, and we will try to use new genes and add phenylalanine to promote the synthesis of 2-PE.

We modeled aroG-fbr homology and aligned the molecular model of the enzyme with the original type:

![](https://static.igem.wiki/teams/4146/wiki/wiki-test/result/fig4.png)

Figure 4. BUCT homology modeling aroG-fbr, original type on the left is blue and BUCT mutant on the middle is pink.

The mechanism by which phenylalanine inhibits AroG activity is very complex. The new AroG binds to Phe and the whole molecule undergoes a dramatic conformational change. There is a study shows that our mutant aroGfbr was shown to alleviate the inhibitory effect of the phenylalanine analogue 4-fluoro-DL-phenylalanine (p-EP). p-EP is a stronger inhibitor of aroG than Phe, and when aroG is inhibited by p-EP, E. coli cannot grow. We visualized this part of the conformational change by homology modeling. We hypothesized that the conformational change of the enzyme protein caused by the mutation might be the main reason for the reduced inhibitory effect of Phe. In the model comparison results, we found that the mutated molecular pocket appears to be covered by a larger lid.

![](https://static.igem.wiki/teams/4146/wiki/wiki-test/result/fig5.png)

Figure 5. The red and dark blue sections represent the molecular conformational changes after the aroG sequence mutation. The spatial position of residues in front of the molecular cavity increases, and the entrance of Phe as an inhibitor into the binding site is like blocked.

# Synthesis of Caffeine

![](https://static.igem.wiki/teams/4146/wiki/wiki-test/result/fig6.jpg)

Figure 6 Caffeine fermentation sampling record

In order to better detect the synthesis of caffeine, the cumulative amount of theobromine was also measured when the fermentation products were measured, and the standard curves were made respectively, as shown in Figure 6 and Figure 7.

![](https://static.igem.wiki/teams/4146/wiki/wiki-test/result/fig7.png)

Figure 7. Standard curve for measurement of theobromine by HPLC

![](https://static.igem.wiki/teams/4146/wiki/wiki-test/result/fig8.png)

Figure 8. Standard curve for measurement of caffeine (CA) by HPLC

The above standard curves are in line with the standard and can be used for the conversion of theobromine and caffeine production. Finally, the maximum yield of theobromine was 7.16mg/L and the maximum yield of caffeine was 1.83mg/L. In the results, we can find theobromine was more productive than caffeine, and it was speculated that the lower methylase activity in the last step resulted in the accumulation of theobromine and affected the synthesis of caffeine. Whether the final output meets our requirements is calculated and forecast through modeling (see **Yield prediction and promoter screening** below).

![](https://static.igem.wiki/teams/4146/wiki/wiki-test/result/fig9.jpg)

Figure 9. Yield of theobromine fermentation in E. coli Nissle 1917

![](https://static.igem.wiki/teams/4146/wiki/wiki-test/result/fig10.jpg)

Figure 10. Yield of caffeine fermentation in E. coli Nissle 1917

# Yield prediction and promoter screening

![](https://static.igem.wiki/teams/4146/wiki/wiki-test/result/fig11.png)

Figure 11. Strain growth fitting curve

In the modeling work, the strain growth fitting curve represents the accuracy of our calculation to a certain extent. And based on that, we predicted caffeine production.

![](https://static.igem.wiki/teams/4146/wiki/wiki-test/result/fig12.png)

Figure 12. Product formation kinetc

By using the Logistic and Luedeking-Piret equations, we have succeeded in constructing a model that can predict caffeine production, and the resulting model fits the experimental data well, proving the feasibility of the model.

It can be concluded that the engineered bacteria are able to produce enough caffeine in practice to exceed the 0.4 mg/L effective concentration threshold. We have also created a model of the strain competition mechanism and used it to assist the design of the suicide mechanism. We calculated that the triggering OD=0.21, at which the strain could suppress Malassezia at various concentrations effectively, while at the same time initiate mazF gene expression, induce apoptosis and ensure that the engineered bacteria do not spread excessively.

We invite you to the [**Model page**](https://2022.igem.wiki/buct/model).

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