iGEM 2020 — Team GZ_HFI

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# Team:GZ HFI/Notebook

Notebook | iGEM GZ\_HFI

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  [Experiment Overview](https://2020.igem.org/Team%3AGZ_HFI/Experiment_Overview)[Measurement](https://2020.igem.org/Team%3AGZ_HFI/Measurement)[Results](https://2020.igem.org/Team%3AGZ_HFI/Results)[Safety](https://2020.igem.org/Team%3AGZ_HFI/Safety)[Notebook](https://2020.igem.org/Team%3AGZ_HFI/Notebook)[Contribution](https://2020.igem.org/Team%3AGZ_HFI/Contribution)
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# Notebook

---

##### Contents

# Construction of ammonia metabolic pathway

1. Construction of plasmids pBR322-KanR-pTac-argA and pBR322-KanR-pTac-argA ^ fbr;

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# Team:GZ HFI/Engineering

Engineering | iGEM GZ\_HFI

[iGEM GZ\_HFI](https://2020.igem.org/Team%3AGZ_HFI)

* Project

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* Experiment

  [Experiment Overview](https://2020.igem.org/Team%3AGZ_HFI/Experiment_Overview)[Measurement](https://2020.igem.org/Team%3AGZ_HFI/Measurement)[Results](https://2020.igem.org/Team%3AGZ_HFI/Results)[Safety](https://2020.igem.org/Team%3AGZ_HFI/Safety)[Notebook](https://2020.igem.org/Team%3AGZ_HFI/Notebook)[Contribution](https://2020.igem.org/Team%3AGZ_HFI/Contribution)
* [Model](https://2020.igem.org/Team%3AGZ_HFI/Model)
* Human Practices

  [Simple Human Practices](https://2020.igem.org/Team%3AGZ_HFI/Simple_Human_Practices)[Integrated Human Practices](https://2020.igem.org/Team%3AGZ_HFI/Integrated_Human_Practices)[Education](https://2020.igem.org/Team%3AGZ_HFI/Education)
* Parts

  [Parts Overview](https://2020.igem.org/Team%3AGZ_HFI/Parts_Overview)[Basic Parts](https://2020.igem.org/Team%3AGZ_HFI/Basic_Parts)[Composite Parts](https://2020.igem.org/Team%3AGZ_HFI/Composite_Parts)[Part Collection](https://2020.igem.org/Team%3AGZ_HFI/Part_Collection)
* [Judging](https://2020.igem.org/Team%3AGZ_HFI/Judging)
* People

  [Team](https://2020.igem.org/Team%3AGZ_HFI/Team)[Collaborations](https://2020.igem.org/Team%3AGZ_HFI/Collaborations)

[ ]

# Engineering

---

##### Contents

# Project Design

## Probiotics Selection

It is important to choose a safe probiotic microorganism because the final product is designed to be orally taken. We selected *E.coli* *Nissle 1917* (*EcN*) as the engineered chassis. *EcN* has been used as probiotics in Europe for a long time in history (Wessenaar, 2016). Moreover, by examining the excretion of healthy volunteers who orally taken *EcN*, researchers found out that the median clearance time of *EcN* is one week, and for 80% of healthy volunteers, the clearance time is less than three weeks (Kurtz, 2018). Thus, *EcN does* not cause long-term harm to the human digestive system.

*EcN* also serves as the agent for probiotic therapy. Various studies have used *E.coli* to treat relapse of ulcerative colitis and bowel disease, which showed positive consequences. Those studies further ensure the biosafety of *EcN* in humans (Kruis, 1917; Rembacken, 1999).

## Construction of the Metabolic Pathways

### Conversion Pathway

The genetic design for ammonia metabolic pathway is inspired by a 2019 research in which the researchers genetically engineered *EcN* strain to convert ammonia to arginine in responding to an intestinal anaerobic environment. It shows a positive result in mice treated with hyperammonemia (Kurtz, 2019). Arginine is a crucial intermediate in the urea cycle, and more importantly, its chemical formula contains four nitrogen, which makes it an ideal option for converting ammonia efficiently (Urea Cycle, n.d.; Arginine, n.d.).

The ammonia that enters into *E.coli* is converted to glutamate along with α-Ketoglutarate (Fig.1). The series of reactions that convert glutamate into arginine are catalyzed by *N*-acetyl glutamate synthetase (NAGS), encoded by gene *argA*, to acetylize glutamate (Cunin, 1986).

However, the arginine synthesis pathway is repressed through two regulatory pathways. First, arginine itself has feedback inhibition on NAGS (Vyas, 1963). In order to maximize the conversion of ammonia to arginine, feedback-resistant (fbr) NAGS enzymes is required, whose *argA* gene is mutated into *argA^fbr* by substituting the sequences TAT (Tyr) at 19th position into TGT (Cys), and the GCG (Ala) at 389th position into GCT (Ala) (Rajagopal, 1998). Second, arginine synthesis is restricted through the arginine regulation, because the formation of crucial enzymes is not only controlled by the conjunction of arginine but also arginine repressor encoded by the regulatory gene *argR* (Lim, 1987). Thus, *argR* is designed to be knocked out in the genome.

![The metabolism pathway of ammonia conversion. The figure is modified from figure 1.A in Ref. Kurtz (2019).](https://static.igem.org/mediawiki/2020/8/8f/T--GZ_HFI--img--design001.png)

In the pathway of converting hydrogen sulfide to L-cysteine, L-serine and Acetyl-CoA form O-acetylserine catalyzed by L-serine O-acetyltransferase (SAT) (Fig. 2). SAT is encoded by gene *cysE*, and its feedback is inhibited by the final product L-cysteine (Kondoh, 2019). In order to induce overproduction of L-cysteine, we need to develop a mutant gene of cysE which will code for feedback inhibition-insensitive SAT. One study suggests turning the ATG (Met) at the 256th position into TGG (Trp) by mutation to obtain *cysE-256* (Nakamori, 1998). Another study created several different *cysE* mutants that overexpress non-feedback-resistant *cysE*, and the best two mutants are selected(Kai Y, 2006), named as *cysE-5*, to substitutes GTC (Val), GAT (Asp) at the 95th and 96th positions for AGA (Arg), CCC (Pro), and *cysE 11-2*, which substitutes CGT (Arg), ACC (Thr) for CAT (His), GTA (Val) at 89th, 90th position, and CCG (Pro), GCA (Ala) for GCT (Ala), ACA (Thr) at 93th and 94th position (Kai, 2006).

To figure out whether there exists a better mutant, new types of *cysE* mutants are created by combining different mutants together (*cysE 5-11-2*; *cysE 256-5*; *cysE 256-11-2*; *cysE 256-5-11-2*).

The efficiency of different mutants to overproduce cysteine is tested and the most optimal mutant is selected into product design.

![The metabolism pathway of hydrogen sulfide conversion. The figure is modified from figure 1 in Ref. Nakatani (2012).](https://static.igem.org/mediawiki/2020/a/a9/T--GZ_HFI--img--design002.png)

Figure 2: The metabolism pathway of hydrogen sulfide conversion. The figure is modified from figure 1 in Ref. Nakatani (2012).

### Myrcene Synthesis Pathway

We also aim to construct the E.coli to produce a fragrant substance to mask the acrid smell. For the purpose of both producing aroma and being safe for humans, we selected terpenes, which can be made naturally in plants, to be the agent masking the acrid smell (Berger, 2007). Myrcene, with the lowest toxicity among the monoterpene family, is a type of terpenes-kind substance that can be naturally produced. Researches also prove that the E.coli pierced with myrcene almost exhibits no growth inhibition (Kim, 2015). Thus, we design to engineer pathways to produce myrcene. Three plasmids are constructed containing essential genes: pMevT, pMBIS, and pTYT-GPPS-MS (Fig. 3). The myrcene pathway MevT contains three genes that encode for necessary enzymes to catalyze the reaction that transforms acetyl-CoA into mevalonate (Fig.3 A). These three genes are AtoB from E.coli and HMGS and HMGR from Saccharomyces cerevisiae (Woo, 2013). MBIS pathway contains five different genes that encode for necessary enzymes to catalyze the reaction that transforms mevalonate into isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), which are ERG12, ERG8, MVD1, idi, and ispA from Saccharomyces cerevisiae (Fig.3 B) (Martin, 2003). Next, IPP and DMAPP are catalyzed by geranyl pyrophosphate synthase (fig.3 B), which is encoded by gene GPPS, to form geranyl diphosphate, the precursor of monoterpene. Then geranyl diphosphate is converted into β-myrcene via myrcene synthase encoded by MS gene (Fig. 3 B) (Kim, 2012). In one study that engineered E.coli to produce myrcene, the researchers test the performance and efficiency of GPPS and MS gene from different kinds of organisms, and from their results, we select the MS(Qi) gene from Quercus ilex L. and GPPS2 from Abies grandies (Kim, 2015).

![The metabolism pathway of myrcene. (A) The MevT pathway. (B) MBIS Pathway and genes GPPS and MS. The figure is modified from figure 1.A in Ref. Kim (2012) and figure 1 in Ref. Matin (2003).](https://static.igem.org/mediawiki/2020/6/69/T--GZ_HFI--img--design003.png)

Figure 3: The metabolism pathway of myrcene. (A) The MevT pathway. (B) MBIS Pathway and genes GPPS and MS. The figure is modified from figure 1.A in Ref. Kim (2012) and figure 1 in Ref. Matin (2003).

### Intestine Homeostasis

In the human digestive system, a delicate bioregulatory system maintains intestinal homeostasis. In the urea cycle, arginine is broken down into urea and ornithine by arginase (Walker, 2014). The gut bacterium, such as *E.coli, Salmonella enterica*, *Clostridia*, and *Enterobacter aerogenes*, can convert cysteine back to hydrogen sulfide (Kumagai, 1975). Human intestinal tissue also conducts the conversion by the cystathionine beta‑synthase and cystathionine gamma‑lyase (Martin, 2010).

Therefore, the natural mechanism of homeostasis will prevent arginine and cysteine from affecting the intestinal balance in the long term. In other words, the engineered *E.coli* would only temporarily lower the concentration of hydrogen sulfide in the intestine.

# Experiment Overview

To both relieve those patients' dilemmas and prevent healthy people from the embarrassment of farting, we intend to genetically engineer probiotic *Escherichia coli* to decline the production of the main molecules that contribute to the acrid smell (ammonia and hydrogen sulfide) and produce an aroma substance (myrcene). We mutated *argA* into *argA^fbr* and knocked out *argR* to enhance the conversion of ammonia into arginine; createed several mutants of *cysE* gene to promote the conversion of hydrogen sulfide into cysteine; and constructed a system of three plasmids containing essential genes to produce myrcene. The results showed that, compared to the control groups, the expression of *argA^fbr* plasmid along with △*argR* genome significantly improved the bacteria's ability to absorb ammonia compared to the control groups. Cysteine synthesis was improved apparently by the expression of mutated *cysE* genes. Besides, engineered *E.coli* produced myrcene successfully but with relatively low production.

# Measurement

## Culture medium

Luria-Bertani (LB) medium (1% Bacto Tryptone, 0.5% Bacto Yeast Extract, and 1% NaCl), M9 medium(1x M9 salts, 1 mM thiamine hydrochloride, 0.4% glucose, 0.2 casamino acids, 2mM magnesium sulfate, and 0.1 mM calcium chloride) was used for general cultivation. If necessary, chloramphenicol (Cm; 30 μg/ml), kanamycin (Kana; 50 μg/ml), ampicillin (Amp; 100 μg/ml) or/and tetracycline (Tet; 10 μg/ml) was added. For solid media, 1.5% (wt/vol) agar was added. As for the arginine synthesis experiment, 5 mM ammonia(aq) was added as an ammonia source; for the cysteine synthesis experiment, 30 mM ammonium sulfate was added as a sulfate source; and 1% glucose was added as an extra carbon source in myrcene synthesis experiment. For all experiments, *EcN* and *DH10b* grew at 37 degree Celsius and 200 rpm in Deep-well Multiwell Plate overnight and test tubes with 0.5 mM isopropyl-β-D-1-thiogalactopyr-anosid(IPTG), except gene knockout which was at 30 degree Celsius.

# Results

## **Strains and plasmids**

### **Construction of arginine maximization plasmids**

The arginine maximization plasmid was constructed by mutating *argA* gene(from *E.coli* strain *DH10b*, encoding for N-acetyl glutamate synthase) into *argAfbr* which substituting the TAT (Tyr) in 19th position into TGT (Thr), and the GCG (Ala) in 389th position into GCT (Ala) through polymerase chain reaction (PCR)). Meanwhile, the original *argA* gene was inserted into the pTYT plasmid(pTYT-argA) as a control group. Besides, CRISPR-cas9 was used to knock out gene *argR* from the genome of *DH10b*. Therefore, *DH10b* with pTYT-argA, pTYT- *argAfbr*, and pTYT-argA-∆argR, pTYT-*argAfbr* -∆argR are constructed along with the wildtype.

Primers argA-F and argA-R (Table.5) were used to obtain the original *argA* gene from the genome by polymerase chain reaction(PCR). GoldenGate was used to connect the PCR product to the pET28B plasmid with promoter *Ptac*. A set of primers argA-Mut-F1, argA-Mut-R2, argA-Mut F2, and argA-Mut R1 (Table.5) were used to mutate the *argA* to *argAfbr* gene, the PCR product was digested by DpnI Restriction Endonuclease and then connected by Gibson assembly. The connection was verified by primers rrnBT-F1 and Ptac-R1 (Table.5) to detect if the genes were successfully inserted.

The upstream and downstream fragments of *argR* gene were obtained by primer sets Up-arm F along with Up-arm R, and down-arm F along with down-arm R respectively (Table.5). The gRNA was designed with the website https://www.atum.bio/eCommerce/cas9/input and was obtained by gRNA-1-F1, gRNA-1-F2, and Scarfold-R (Table.5). The gRNA, upstream and downstream fragments were constructed into pTarget using Gibson assembly.

The constructed pTarget plasmid along with pCas plasmid was transformed into both *E.coli EcN* and *E.coli DH10b* to delete *argR* gene.

**Table.4** **The list of all stains and plasmid used.** *The relevant characteristics of plasmids are listed in the following order: type of replication origin, antibiotic resistance, type of promoter, standard plasmid name.*

|  |  |  |
| --- | --- | --- |
| strain or plasmid | relevant characteristics | reference |
| strains |  |  |
| E.coli DH5a | F-80d lacZ∆M15 ∆(lacZYA-argF) U169 end A1 recA1 hsdR17 (rk-, mk+) supE44– thi-1 gtrA96 relA1 phoA | Ref. Trans5α Chemically Competent Cell, n.d. |
| E.coli EcN | E.coli Nissle 1917 |  |
| E.coli DH10b | F- mcrA ∆(mrr-hsdRMS-mcrBC) 80 lacZ∆M15∆lacX74 recA1 ara∆139∆(ara-leu)7697 galU galK rpsL (StrR) endA1 nupG | Ref. Trans10 Chemically Competent Cell, n.d. |
| E.coli DH10b ∆argR | F- mcrA ∆(mrr-hsdRMS-mcrBC) 80 lacZ∆M15∆lacX74 recA1 ara∆139∆(ara-leu)7697 galU galK rpsL (StrR) endA1 nupG ∆argR | this work |
| plasmid for arginine synthesis |  |  |
| pTYT-argA | pUC, Kanar, Ptac, pET28B-argA | this work |
| pTYT-argAfbr | pUC, Kanar, Ptac, pET28B-argA^fbr | this work |
| plasmid for cysteine synthesis |  |  |
| pTYT-cysE | pUC, Kanar, Ptac, pET28B-cysE | this work |
| pTYT-cysE-256 | pUC, Kanar, Ptac, pET28B-mut cysE 256 | this work |
| pTYT-cysE-5 | pUC, Kanar, Ptac, pET28B-mut cysE 5 | this work |
| pTYT-cysE-11-2 | pUC, Kanar, Ptac, pET28B-mut cysE 11-2 | this work |
| pTYT-cysE-5-11-2 | pUC, Kanar, Ptac, pET28B-mut cysE 5&11-2 | this work |
| pTYT-cysE-256-5 | pUC, Kanar, Ptac, pET28B-mut cysE 256&5 | this work |
| pTYT-cysE-256-11-2 | pUC, Kanar, Ptac, pET28B-mut cysE 256&11-2 | this work |
| pTYT-cysE-256-5-11-2 | pUC, Kanar, Ptac, pET28B-mut cysE 256&5&11-2 | this work |
| plasmid for myrcene synthesis |  |  |
| pTYT-GPPS-MS | pUC, Kanar, Ptac, pET28B-tGPPS-MS\_Qi | this work |
| pMevT | p15A, Cmr, Plac, pMBIS-atoB-HMGS-tHMGR | Jay Keasling's engineered Saccharomyces cerevisiae |
| pMBIS | pBBR1, Tetr, Plac , pMBIS-ERG12-ERG8-MVD1-idi-ispa | Jay Keasling’s engineered Saccharomyces cerevisiae |
| pTarget | pUC, Ampr, J23119, pTarget-upArm-gRNA-downarm | this work |
| pCas9 | pSC101, Kanar, Pcas, pCas-cas9-gam-bet | Ref. Jiang, 2015 |

### **Construction of cysteine maximization plasmids**

Three mutants in *cysE* gene were utilized and inserted into the following plasmids: pTYT-cysE-256 to express the mutant *cysE-256*(substitute the ATG (Met) on the 256th position into TGG (Trp)), pTYT-cysE-5 to express the mutant *cysE-5* (mutate the GTC (Val), GAT (Asp) on the 95th and 96th position into AGA (Arg), CCC (Pro)), and pTYT-cysE-11-2 to express the mutant *cysE-11-2*(substitutes CGT (Arg), ACC (Thr) in 89th, 90th positions into CAT (His), GTA (Val)), and substitutes CCG (Pro), GCA (Ala) in 93th and 94th position into GCT (Ala), ACA (Thr)). By combining these mutants, more mutants were created: plasmid pTYT-cysE-256-5(combine the

positions of mutations in mutants *cysE-256* and *cysE-5*), pTYT-cysE-256-11-2(combine the positions of mutations in mutants *cysE-256* and *cysE-11-2*), pTYT-cysE-256-5-11-2(combine the positions of mutations in mutants *cysE-256*, *cysE-5*, and *cysE-11-2*), and pTYT-cysE-5-11-2 (combine the positions of mutations in mutants *cysE-5* and *cysE-11-2*) were constructed on the pET28B plasmids. Meanwhile, original *cysE* was constructed on the same plasmid as pTYT-cysE with the same promoter served as the control along with wildtype (Table.4).

The original *cysE* gene was obtained from the genome in *DH5a* by PCR with primers cysE-F and cysE-R (Table.5). GoldenGate was used to connect gene *cysE* to plasmid pET28B to construct pTYT-cysE. Primers cysE-mut-F1, cysE-Mut-R2, cysE-Mut R1, and cysE-Mut-F2 were used to mutate *cysE* to *cysE-256* with templated pTYT-cysE, which was digest by DpnI Restriction Endonuclease (Table.5), then connected by overlapped PCR to construct the plasmid pTYT-cysE-256. Similarly, primers cysE5&11-Mut-F1, cysE5&11-Mut-R2, cysE5-mut-F1, cysE5-mut-R2, cysE11-mut-F1, and cysE11-mut-R2 were used to obtain genes *cysE-5*, *cysE-11-2*, *cysE-5-11-2*, *cysE-256-5*, *cysE-256-11-2*, and *cysE-256-5-11-2* by PCR with templates pTYT-cysE and pTYT-cysE-256 (Table.6), which were digested by DpnI, and connected by overlap PCR to pET28B plasmid. These were verified by PCR with primers cysE-JC-F and cysE-JC-R if the mutation was gained, and rrnBT-F1 and Ptac-R1 if the genes were inserted into the pET28B plasmid (Table.5).

**Table. 5 Information on primers used in this research**

| primer | function | sequence |
| --- | --- | --- |
| argA-F | Obtain the argA gene. | 5'-TGGAATTCGCGGCCGCTTCTAGAGGTGGTAAAGGAACGTAAAACCG-3' |
| argA-R | Obtain the argA gene. | 5'-GGACTGCAGCGGCCGCTACTAGTATTACCCTAAATCCGCCATCAA-3' |
| argA-Mut-F1 | Mutate the argA to argAfbr gene | 5'-TTCCCTGTATCAATACCCACCG-3' |
| argA-Mut-R2 | Mutate the argA to argAfbr gene | 5'-CGGTGGGTATTGATACAGGGAA-3' |
| argA-Mut-F2 | Mutate the argA to argAfbr gene | 5'-TCAGGCTAAGCAGAGC-3' |
| argA-Mut-R1 | Mutate the argA to argAfbr gene | 5'-GCTCTGCTTAGCCTGA-3' |
| cysE-F | Obtain the cysE gene. | 5'-TGGAATTCGCGGCCGCTTCTAGAGATGTCGTGTGAAGAACTGGAA-3' |
| cysE-R | Obtain the cysE gene. | 5'-CGCTACTAGTATTAGATCCCATCCCCATACTC-3' |
| pTarget-JC-F | Verify the connection of up-Arm down-Arm and gRNA | 5'-TTGCTGGCCTTTTGCTCACATG-3' |
| pTarget-JC-R | Verify the connection of up-Arm down-Arm and gRNA | 5'-TCGATCATAGCACGATCAACGGC-3' |
| gRNA-1-F2 | Construct gRNA sequence | 5'-CAGTCCTAGGTATAATACTAGTAGAAGAGAAATTTAGCTCCCGTTTTAG-3' |
| gRNA-1-F1 | Construct gRNA sequence on pTarget | 5'-TAATACTAGTAGAAGAGAAATTTAGCTCCCGTTTTAGAGCTAGAAATAGCAAGTTAAAA-3' |
| Scarfold-R | Construct and connect gRNA sequence on pTarget | 5'-CTGCAGGTCGACTCTAGAGA-3' |
| pTarget-VR | Construct pTarget plasmid vector | 5'-ACTAGTATTATACCTAGGACTGAGCT-3' |
| pTarget-VF | Construct pTarget plasmid vector | 5'-AAGCTTAGATCTATTACCCTGTTATCC-3' |
| UP-Arm-F | Obtain and construct up-Arm gene from genome | 5'-TCTCTAGAGTCGACCTGCAGGGTTTTTAACAGTAGTGCAAGCGC-3' |
| UP-Arm-R | Obtain and construct up-Arm gene from genome | 5'-AAGTCACCCGATATGGTGGTTG-3' |
| DOWN-Arm-F | Obtain and construct up-Arm gene from genome | 5'-ACCACCATATCGGGTGACTTTCTCTGCCCCGTCGCTTCTG-3' |
| DOWN-Arm-R | Obtain and construct up-Arm gene from genome | 5'-CAGGGTAATAGATCTAAGCTTGCCACACCACTTACGGATACG-3' |
| rrnBT1-R1 | Verify gene on pTYT | 5'-TGCGCCGCTACAGGGCGCGTGAGAGCGTTCACCGACAAACAA-3' |
| Ptac-F1 | Verify gene on pTYT | 5'-AGATCTCGATCCCGCGAAATTTCGTCAGGCCACATAGCTT-3' |
| MS-F | Obtain the MS gene and connect to GPPS | 5'-TACTAGAGAAAGAGGAGAAATACTAGATGCGAAGAAGCGCGAATTATCA-3' |
| MS-R | Obtain the MS and connect to pTYT | 5'-TCGTTTTATTTGATGCCTGGACTAGTATTAGTCCTTGTTCAGCGGGA-3' |
| GPPS-F | Obtain the GPPS and connect to pTYT | 5'-AAACAGCCTCTACAAATAATTTTGTTTAAATACCCGTTTTTTGGGCTAA-3' |
| GPPS-R | Obtain the GPPS gene and connect to MS | 5'-CTAGTATTTCTCCTCTTTCTCTAGTATTATTTGCTGCGTTTGTAAACCT-3' |
| cysE-Mut-F1 | Mutate cysE to cysE-256 | 5'-CGCTAAGGATGATTTCTGGAATTCGC-3' |
| cysE-Mut-R2 | Mutate cysE to cysE-256 | 5'-GCGAATTCCAGAAATCATCCTTAGCGAAAG-3' |
| cysE-Mut-R1 | Mutate cysE to cysE-256 | 5'-GTTGAAATGCTGGTCCCAATCCATTGATGG-3' |
| cysE-Mut-F2 | Mutate cysE to cysE-256 | 5'-CATCAATGGATTGGGACCAGCATTTCAAC-3' |
| cysE5&11-Mut-F1 | Mutate cysE to cysE-5-11-2 & cysE-256-5-11-2 | 5'-CATGTACGCGACGCTACAAGACCCAAATACTCAACCCCGTTG-3' |
| cysE5&11-Mut-R2 | Mutate cysE to cysE-5-11-2 & cysE-256-5-11-2 | 5'-CTTGTAGCGTCGCGTACATGCACCGCCTGAATATCACAGG-3' |
| cysE5-mut-F1 | Mutate cysE to cysE-5 & cysE-256-5 | 5'-CCCGCGACCCGGCAAGACCCAAATACTCAACCCCGTTGTTAT-3' |
| cysE5-mut-R2 | Mutate cysE to cysE-5 & cysE-256-5 | 5'-GGGTCTTGCCGGGTCGCGGGTACGCAC-3' |
| cysE11-mut-F1 | Mutate cysE to cysE-11-2 & cysE-256-11-2 | 5'-TGCATGTACGCGACGCTACAGTCGATAAATACTCAACCCCGTTGT-3' |
| cysE11-mut-R2 | Mutate cysE to cysE-11-2 & cysE-256-11-2 | 5'-TGTAGCGTCGCGTACATGCACCGCCTGAATATCACAGG-3' |
| cysE-JC-F | Verify the mutation of cysE | 5'-TGCTGGCGAACAAGCTGTCATC-3' |
| cysE-JC-R | Verify the mutation of cysE | 5'-GGAACGTCACAGAAACCTGGTTTTG-3' |
| genone-F | Verify the gene knockout | 5'-CTGGAGCGATATCATACAGAGAGAGTTC-3' |
| genone-R | Verify the gene knockout | 5'-GTTCAGCATTTCACGCATATCCATTGGC-3' |

**Table.6 Designed mutations on plasmids used in this research.**

| Allele of the cysE gene | sequence of proteins and cysE gene at position 89-96 | SAT protein and cysE gene at position 254-256 |
| --- | --- | --- |
| cysE wild-type | Arg Thr Arg Asp Pro Ala Val AspCGT ACC CGC GAC CCG GCA GTC GAT | Met Asp Met Asp GlnATG GAT ATG GAC CAG |
| cysE 256 | Arg Thr Arg Asp Pro Ala Val AspCGT ACC CGC GAC CCG GCA GTC GAT | Met Asp Trp Trp GlnATG GAT TGG GAC CAG |
| cysE 5 | Arg Thr Arg Asp Pro Ala Arg ProCGT ACC CGC GAC CCG GCA AGA CCC | Met Asp Met Asp GlnATG GAT ATG GAC CAG |
| cysE 11-2 | His Val Arg Asp Ala Thr Val AspCAT GTA CGC GAC GCT ACA GTA GAT | Met Asp Met Asp GlnATG GAT ATG GAC CAG |
| cysE 5-11-2 | His Val Arg Asp Ala Thr Arg ProCAT GTA CGC GAC GCT ACA AGA CCC | Met Asp Met Asp GlnATG GAT ATG GAT CAG |
| cysE 256-5 | Arg Thr Arg Asp Pro Ala Arg ProCGT ACC CGC GAC CCG GCA AGA CCC | Met Asp Trp Trp GlnATG GAT TGG GAC CAG |
| cysE 256-11-2 | His Val Arg Asp Ala Thr Val AspCAT GTA CGC GAC GCT ACA GTA GAT | Met Asp Trp Trp GlnATG GAT TGG GAC CAG |
| cysE 256-5-11-2 | His Val Arg Asp Ala Thr Arg ProCAT GTA CGC GAC GCT ACA AGA CCC | Met Asp Trp Trp GlnATG GAT TGG GAC CAG |

### **Construction of myrcene production plasmids**

Two engineered *Saccharomyces cerevisiae* plasmids pMevT and pMBIS constructed by Jay Keasling (originally used to produce arteannuic acid) were provided. The pMevT plasmid contains three genes *AtoB*(from *E.coli*), *HMGS*(from *Saccharomyces cerevisiae*), and *tHMGR*(from *Saccharomyces cerevisiae*) with promoter *Plac* (Table.4). The pMBIS plasmid contains five genes, which are *ERG12*(from *Saccharomyces cerevisiae*), *ERG8*(from *Saccharomyces cerevisiae*), *MVD1*(from *Saccharomyces cerevisiae*), *idi*(from *Saccharomyces cerevisiae*), and *ispA*(from *Saccharomyces cerevisiae*), with promoter *Plac*. The third plasmid pTYT-GPPS-MS was constructed and it contains genes *GPPS*(from *Abies Grandis*, position 1-84) and *MS*(from *Quercus ilex L*, position 1-56). They were constructed on the pET28B substituting promoter *PT7* to *Ptac*.

pMevT and pMBIS were obtained from Jay Keasling's laboratory. *GPPS* was obtained from plasmid pLB1s-Erg20(M) provided by biotech company *Bluepha* with primers GPPS-F and GPPS-R, and *MS* was obtained from synthesis plasmid with primer MS-F and MS-R provided by biotech company *GenScript*. The pET28B vector was linearized with BsaI. The *GPPS* fragment, *MS* fragment, and the linearized vector were connected by overlap PCR with primer GPPS-F and MS-R to construct pTYT-GPPS-MS. Primers rrnBT-F1 and Ptac-R1 were designed to detect if the genes are inserted into the pET28B plasmid(table.5).

## **Measurement of ammonia**

### **Reagent preparation**

Nessler's reagent was prepared by weighing 16 g sodium hydroxide (NaOH), dissolving with 50 ml distilled water, and waiting until it is cold as the surrounding. 7 g potassium iodide and 10 g mercury iodide are dissolved consecutively into the solution. The solution then is poured into the aqua of sodium hydroxide slowly while stirring and diluted into 100 ml.

Sodium potassium tartrate tetrahydrate reagent is prepared by weighing 50 g sodium potassium tartrate tetrahydrate dissolving with 100 ml distilled water.

Besides, the molarities of standard samples which mixed ammonium sulfate with distilled water are 0.025mM, 0.05mM, 0.1mM, 0.2mM, 0.4mM, 0.5mM.

### **Determination of Calibration line**

96-well plates were used added with 150 μL samples, 30 μL sodium potassium tartrate tetrahydrate reagent, and 20 μL Nessler's reagent in turn. After 10 minutes of waiting, *infinite 200Pro* spectrophotometer was used to test the light absorption at 420 nm. A reagent blank without ammonium sulfate was prepared under the same condition. The least-squares regression line was drawn according to data collected. The x-axis refers to the absorbance of light at a certain wavelength, and the y-axis refers to the concentration of NH4+ in solution.

### **NH4+ analysis and quantification**

Before the experiment, *DH10b* was cultivated in M9 medium with 5mM ammonia and 0.1 mM IPTG in Deep-well Multiwell Plate at 37 degree Celsius overnight. 10 μL supernatant of each medium was taken from Deep-well Multiwell Plate and diluted with 990 μL distilled water in 1.5 mL centrifuge tubes respectively and mixed thoroughly. The reagent blank with only pure M9 medium was prepared under the same condition. The same operation was repeated to test standard samples, and then used spectrophotometer with light absorption at 420 nm to test the concentration of ammonia in the solutions.

## **Measurement of cysteine**

### **Reagent preparation**

The acid ninhydrin reagent contained 250mg of ninhydrin in a mixture of 6ml of acetic acid and 4 ml of conc. HCl, which was mixed repeatedly at room temperature, yielded a solution in 20 - 30 min. This reagent was prepared before use. Besides, the molarities of standard samples which mixed solid L-cysteine with distilled H2O are 0.1mM, 0.2mM, 0.4mM, 0.8mM and 1mM respectively (Gaitonde, 1967).

### **Determination of Calibration Line**

Centrifuge tubes are added 0.1mM, 0.2mM, 0.4mM, 0.8mM, and 1mM to 1.5 ml standard samples of L-cysteine respectively with 0.1 ml acid ninhydrin reagent and 0.1 ml acetic acid, mixed thoroughly at room temperature. The centrifuge tubes are heated in the metal bath with 100 degrees Celsius for 10 minutes, and then rapidly cool in ice. Then 0.7 ml 95% ethanol is added in each centrifuge tubes and mixed thoroughly. A reagent blank without cysteine is prepared under the same condition. The pipette is used to shift 200 microliter solutions of each mixture to 96-well plates so that solution can be tested by *infinite 200Pro* spectrophotometer, with light absorption at 560 nm. The least-squares regression line is created according to the data collected by the spectrophotometer. The x-axis refers to the absorbance of light at a certain wavelength, and the y-axis refers to the concentration of cysteine in solution.

### **Cysteine analysis and quantification**

Before the test, *EcN* was cultivated in M9 medium with 0.5 mM IPTG in Deep-well Multiwell Plate at 37 degree Celsius overnight. 0.1 ml supernatant of each *EcN* solution is taken from Deep-well Multiwell Plate and added to 1.5 ml centrifuge tubes respectively. The same operation is repeated as testing standard samples, and then use spectrophotometer with light absorption at 560 nm to test the concentration of cysteine in the solutions.

## **Measurement of myrcene**

### **Myrcene purification**

Before the experiment, *DH10b* is introduced in 2 ml M9 medium with 0.5 mM IPTG and 1% glucose, in 37 degree Celsius overnight. 20% dodecane (v/v) was added to collect the myrcene produced by *DH10b*. After the centrifugation (12000rpm for a minute), the dodecane layer was collected to test the production of myrcene (Kim, E. et al, 2015).

### **Myrcene analysis and quantification**

After the dodecane layer was collected, the collected sample was analyzed by gas chromatography−mass spectrometry [GC−MS, Agilent 6890N series GC/TOF-MS(LECO), under these conditions: injector temperature, 250°C; flowrate, 1.2 mL/min; split ratio, 2:1; oven initially at 60°C for 5 min, followed by 4°C/min, and increase to 240°C; carrier gas, N2; and HP-Ultra2 column (25 m long, 0.2 mm diameter, and 0.11μm film thick)]. 95% myrcene sample was used as a standard for quantitative analysis, and the concentrations were normalized using an internal standard.

## Reference

[1] American Cancer Society. (2011). *Global cancer facts & Figures 2nd edition* 14-01-2015.

[2] Berger RG: Flavours and Fragrances: Chemistry, Bioprocessing and Sustainability. *Springer;* 2007.

[3] Cunin R, et al. Biosynthesis and metabolism of arginine in bacteria. *Microbiol Rev* 1986;50(3):314-52.

[4] Dehong YU. Enterostomal therapy in China:status and advances. *Chin J Nurs* 2015;*40*(006), 415-417.

[5] Deng YF, et al. Prebiotics mitigate in vitro sulfur-containing odour generation in caecal content of pigs. *Ital J Anim Sci* 2015;14(1):2015-3762\*.\*

[6] Diébold L, et al. Shame and specular image: experience of colostomy in colorectal cancer. *Psycho-Oncologie* 2016;10(2):128-35.

[7] Edwards BK, et al. Annual report to the nation on the status of cancer, 1975--2006, featuring colorectal cancer trends and impact of interventions (risk factors, screening, and treatment) to reduce future rates. *Cancer 2*010; 116: 544-73.

[8] Gaitonde MK. A spectrophotometric method for the direct determination of cysteine in the presence of other naturally occurring amino acids. *Biochem J*. 1967;104(2):627-633.

[9] Gastinger I, et al. Protective defunctioning stoma in low anterior resection for rectal carcinoma. *Br J Surg* 2005; 92: 1137-42.

[10] Gemert LJV. Compilations of odour threshold values in air, water and other media; *Boelens Aroma Chemical Information Service*, 2003.

[11] Hunde A, et al. Supplementation of poultry feeds with dietary zinc and other minerals and compounds to mitigate nitrogen emissions-a review. *Biol Trace Elem Res* 2012;147(1-3):386-94.

[12] Jiang Y, et al. Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. *Appl Environ Microbiol* 2015;81(7):2506-14.

[13] Kai Y, et al. Engineering of Escherichia coli L-serine O-acetyltransferase on the basis of crystal structure: desensitization to feedback inhibition by L-cysteine. *Protein Eng Des Sel* 2006;19(4):163-7.

[14] Kim EM, et al. Microbial Synthesis of Myrcene by Metabolically Engineered Escherichia coli. *J Agric Food Chem* 2015,13;63(18):4606-12.

[15] Kondoh M, et al. L-Cysteine production by metabolically engineered Corynebacterium glutamicum. *Appl Microbiol Biotechnol* 2019;103(6):2609-19.

[16] Kruis W, et al. Double-blind comparison of an oral Escherichia coli preparation and mesalazine in maintaining remission of ulcerative colitis. *Aliment Pharmacol Ther* 1997;11(5):853-8.

[17] Kumagai H, et al. Crystallization and properties of cysteine desulfhydrase from Aerobacter aerogenes. *FEBS Lett* 1975;52(2):304-7.

[18] Kurtz C, et al. Translational Development of Microbiome-Based Therapeutics: Kinetics of E. coli Nissle and Engineered Strains in Humans and Nonhuman Primates. *Clin Transl Sci* 2018;11(2):200-7.

[19] Kurtz CB, et al. An engineered *E. coli* Nissle improves hyperammonemia and survival in mice and shows dose-dependent exposure in healthy humans. *Sci Transl Med* 2019;11(475):eaau7975.

[20] Levitt MD. Volume and compo­ sition of human intestinal gas. *N Engl J Med* 1971;284(25):1394-8.

[21] Lim DB, et al. Nucleotide sequence of the argR gene of Escherichia coli K-12 and isolation of its product, the argininerepressor. *Proc Natl Acad Sci USA* 1987;84(19):6697-701.

[22] Lynch BM, et al. Stoma surgery for colorectal cancer: A population-based study of patient concerns. *J Wound Ostomy Continence Nurs* 2008;35(4):424-428

[23] Martin VJJ, et al. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids.*Nat Biotechnol* 2003;21(7):796-802.

[24] Matin GR, et al. Hydrogen sulphide synthesis in the rat and mouse gastrointestinal tract. *Dig Liver Dis* 2010;42(2):103-9.

[25] Nakamori S, et al. Overproduction of L-Cysteine and L-Cystine by *Escherichia coli* Strains with a Genetically Altered Serine Acetyltransferase. *Appl Environ Microbiol* 1998; 64(5):1607-11.

[26] Nakatani T, et al. Enhancement of thioredoxin: glutaredoxin- mediated L-cysteine synthesis from S-sulfocysteine increases L-cysteine production in Escherichia coli. *Microb Cell Fact* 2012;11:62.

[27]Rajagopal BS, et al. Use of Inducible Feedback-Resistant *N*-Acetylglutamate Synthetase (*argA*) Genes for Enhanced Arginine Biosynthesis by Genetically Engineered *Escherichia coli* K-12 Strains. *Appl Environ Microbiol* 1998;64(5):1805-11.

[28] Rembacken BJ, et al. Non-pathogenic Escherichia coli versus mesalazine for the treatment of ulcerative colitis: a randomised trial. *Lancet* 1999;354(9179):635-9.

[39] Urea Cycle. *NYU School of Medicine.* <http://education.med.nyu.edu/mbm/aminoAcids/>

ureaCycle.shtml. Accessed September 13, 2020.

[30] Vonk-Klaassen SM, et al. Ostomy-related problems and their impact on quality of life of colorectal cancer ostomates: a systematic review. Qual Life Res 2016; 25: 125-33.

[31] Vyas S, et al. Feedback inhibition of acetylglutamate synthetase by arginine in Escherichia coli. *Arch Biochem Biophys* 1963; 100(3), 542-6.

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# Team:GZ HFI/Experiments

P-Erfume

[P-Erfume](/)

Project  Project

* [Background](/1_Project/Background.html)
* [Design](/1_Project/Design.html)

Experiment  Experiment

* [Overview](/2_Experiment/Overview.html)
* [Method](/2_Experiment/Method.html)
* [Results](/2_Experiment/Results.html)
* [Safety](/2_Experiment/6_Safety.html)
* [Notebook](/2_Experiment/9_Notebook.html)

[Modeling](/4_Modeling.html)

Human Practices  Human Practices

* [HP](/5_Human_Practices/HP.html)
* [Integrated Human Practices](/5_Human_Practices/Integrated_Human_Practice.html)
* [Public Education](/5_Human_Practices/Public_Education.html)

[Contributions](/7_Contribution.html)

Parts  Parts

* [Overview](/8_Parts/Overview.html)
* [Basic Parts](/8_Parts/Basic_Parts.html)
* [Composite Parts](/8_Parts/Composite_Parts.html)
* [Part Collection](/8_Parts/Part_Collection.html)

[Judging](/10_Judging.html)

People  People

* [Our Team](/11_People/Our_team.html)
* [Collarborations](/11_People/Collaboration.html)
* [Attributions](/11_People/Attributions.html)
* [Gallery](/11_People/Gallery.html)

Project  Project

* [Background](/1_Project/Background.html)
* [Design](/1_Project/Design.html)

Experiment  Experiment

* [Overview](/2_Experiment/Overview.html)
* [Method](/2_Experiment/Method.html)
* [Results](/2_Experiment/Results.html)
* [Safety](/2_Experiment/6_Safety.html)
* [Notebook](/2_Experiment/9_Notebook.html)

[Modeling](/4_Modeling.html)

Human Practices  Human Practices

* [HP](/5_Human_Practices/HP.html)
* [Integrated Human Practices](/5_Human_Practices/Integrated_Human_Practice.html)
* [Public Education](/5_Human_Practices/Public_Education.html)

[Contributions](/7_Contribution.html)

Parts  Parts

* [Overview](/8_Parts/Overview.html)
* [Basic Parts](/8_Parts/Basic_Parts.html)
* [Composite Parts](/8_Parts/Composite_Parts.html)
* [Part Collection](/8_Parts/Part_Collection.html)

[Judging](/10_Judging.html)

People  People

* [Our Team](/11_People/Our_team.html)
* [Collarborations](/11_People/Collaboration.html)
* [Attributions](/11_People/Attributions.html)
* [Gallery](/11_People/Gallery.html)

   ![hero](/hero.png)

# An Innovation to Break the Fart Barrier

Engineering probiotics to reduce intestinal undesirable odor.

[Getting Started](/1_Project/Background.html)

***\*The pronunciation of the letter "P" means "fart" in Chinese. "Perfume" means fragrance***

## [#](#current-problems) Current Problems

* The unpleasant odor of farting leads to social embarrassment.
* Patients who undergo enterostomy are confronting social distancing because of the acrid smell released from the stoma on their bellies.

## [#](#our-contribution) Our Contribution

To solve the problems above, we genetically engineered probiotic Escherichia coli to decrease the production of main molecules that contribute to intestinal acrid smell (ammonia and hydrogen sulfide) and produce an aroma substance (myrcene). We mutate argA into argAfbr and knockout argR to enhance the conversion of ammonia into arginine, create several mutants of cysE gene to promote the conversion of hydrogen sulfide into cysteine, and construct a three-plasmids-system to produce myrcene. The result shows we significantly improved e.coli's ammonia production and cysteine synthesis; myrcene was produced successfully in relatively low production.

For human practices, we did a series of activities (survey, expert visits, collaboration, exhibitions, lectures, etc) to improve our product design and promote the education of synthetics biology.

## [#](#project-promotion-video) Project Promotion Video

## [#](#table-of-contents) Table of Contents

* **Project**
  + [Background](/1_Project/Background.html)
  + [Design](/1_Project/Design.html)
* **Experiment**
  + [Demonstration](/2_Experiment/Overview.html)
  + [Method](/2_Experiment/Method.html)
  + [Results](/2_Experiment/Results.html)
  + [Safety](/2_Experiment/6_Safety.html)
  + [Notebook](/2_Experiment/9_Notebook.html)
* [**Modeling**](/4_Modeling.html)
* **Human Practices**
  + [HP](/5_Human_Practices/HP.html)
  + [Integrated Human Practices](/5_Human_Practices/Integrated_Human_Practice.html)
  + [Public Education](/5_Human_Practices/Public_Education.html)
* [**Contributions**](/7_Contribution.html)
* **Parts**
  + [Overview](/8_Parts/Overview.html)
  + [Basic Parts](/8_Parts/Basic_Parts.html)
  + [Composite Parts](/8_Parts/Composite_Parts.html)
  + [Part Collection](/8_Parts/Part_Collection.html)
* [**Judging**](/10_Judging.html)
* [**People**](/11_People/Our_team.html)

---

**🚡 P-Erfume Wiki** ©2020 Team GZ\_HFI. .

Authored and maintained by Team GZ\_HFI.

# Experiments

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).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

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# Team:GZ HFI/Results

Results | iGEM GZ\_HFI

[iGEM GZ\_HFI](https://2020.igem.org/Team%3AGZ_HFI)

* Project

  [Background](https://2020.igem.org/Team%3AGZ_HFI/Background)[Design](https://2020.igem.org/Team%3AGZ_HFI/Design)[Implementation](https://2020.igem.org/Team%3AGZ_HFI/Implementation)[Proof Of Concept](https://2020.igem.org/Team%3AGZ_HFI/Proof_Of_Concept)
* Experiment

  [Experiment Overview](https://2020.igem.org/Team%3AGZ_HFI/Experiment_Overview)[Measurement](https://2020.igem.org/Team%3AGZ_HFI/Measurement)[Results](https://2020.igem.org/Team%3AGZ_HFI/Results)[Safety](https://2020.igem.org/Team%3AGZ_HFI/Safety)[Notebook](https://2020.igem.org/Team%3AGZ_HFI/Notebook)[Contribution](https://2020.igem.org/Team%3AGZ_HFI/Contribution)
* [Model](https://2020.igem.org/Team%3AGZ_HFI/Model)
* Human Practices

  [Simple Human Practices](https://2020.igem.org/Team%3AGZ_HFI/Simple_Human_Practices)[Integrated Human Practices](https://2020.igem.org/Team%3AGZ_HFI/Integrated_Human_Practices)[Education](https://2020.igem.org/Team%3AGZ_HFI/Education)
* Parts

  [Parts Overview](https://2020.igem.org/Team%3AGZ_HFI/Parts_Overview)[Basic Parts](https://2020.igem.org/Team%3AGZ_HFI/Basic_Parts)[Composite Parts](https://2020.igem.org/Team%3AGZ_HFI/Composite_Parts)[Part Collection](https://2020.igem.org/Team%3AGZ_HFI/Part_Collection)
* [Judging](https://2020.igem.org/Team%3AGZ_HFI/Judging)
* People

  [Team](https://2020.igem.org/Team%3AGZ_HFI/Team)[Collaborations](https://2020.igem.org/Team%3AGZ_HFI/Collaborations)

[ ]

# Results

---

##### Contents

# **Construction of plasmids and strains**

### **Construction of argA plasmids**

To maximize the arginine production, plasmids pTYT-argA and pTYT-argA^fbr are constructed to express *argA* and *argA^fbr* respectively under the control of promoter Ptac which can be induced by IPTG by GoldenGate assembly (fig.7 A). Plasmids pTYT-argA and pTYT-argAfbr were successfully constructed according to the PCR verification (fig. 8).

### **Construction of cysE plasmids**

In order to maximize the cysteine production, plasmids pTYT-cysE, pTYT-cysE-256, pTYT-cysE-5, pTYT-cysE-11-2, pTYT-cysE-5-11-2, pTYT-cysE-256-5, pTYT-cysE-256-11-2, pTYT-cysE-256-5-11-2 were constructed to express *cysE* genes with mutants at position 256, 5 and 11-2 respectively under the control of IPTG-induced promoter Ptac by GoldenGate assembly (fig.7 B). Plasmids pTYT-cysE, pTYT-cysE-256, pTYT-cysE-5, pTYT-cysE-11-2, pTYT-cysE-5-11-2, pTYT-cysE-256-5, pTYT-cysE-256-5-11-2 were verified by PCR as successful constructions (fig. 8), except pTYT-cysE-256-11-2, which was failed to be constructed after several trials.

### **Construction of myrcene production plasmids**

Three plasmids were constructed to produce myrcene in the *E.coli DH10b*. Among them, pMevT and pMBIS were extracted from Jay Keasling's engineered *Saccharomyces cerevisiae* strain to produce arteannuic acid. pMevT contains promoter Plac and genes *AtoB*, *HMGS*, *tHMGR* (fig.7). pMBIS contains promoter Plac and genes *ERG12, ERG8, MVD1, idi, ispa* (fig.7 C). Plasmid pTYT-GPPS-MS was constructed to express genes *GPPS* and *MS* under the control of promoter Ptac by overlap PCR and Gibson assembly (fig.7 C). The successful construction of pTYT-GPPS-MS was verified by PCR (fig. 8).

![](https://static.igem.org/mediawiki/2020/2/2c/T--GZ_HFI--img--results007.png)

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