iGEM 2024 — Team scut-china-a


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Evolution of Halomonas TD

Domestication of acetate tolerance

Introduction of formate assimilation pathway

T-switch

Products

P34HB

Amino acid derivatives：Tyrian Purple

Proteins：SOD and phaP

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# ![flower](https://static.igem.wiki/teams/5226/general/before-h1-2.png)Evolution of *Halomonas* TD

## Domestication of acetate tolerance

#### First cycle: Adaptive laboratory evolution

**Design:**
Acetate is the main product obtained through electrochemical catalytic conversion of CO(2).**The alkaline and halophilic characteristics** of *Halomonas* TD make it highly tolerant to acetate and give it a significant inherent advantage in utilizing acetate as a carbon source, which meets our requirements perfectly. However, *Halomonas* TD01 shows lower tolerance and utilization of acetate compared to glucose. When grown in a minimal medium containing up to 75 g/L sodium acetate (NaAc), the cell growth rate of *Halomonas* TD01 decreases by 56.14%. [1]

**Build:**
ALE of *Halomonas* TD1.0 was performed at 37°C using **50MMA agar plates** supplemented with NaAC. Initially, single colony was isolated on a 60LB agar plate, and then spread on the 50MMA agar plate, followed by incubation at 37°C for 48 h. Then the single colony with optimal cell growth on the 50MMA agar plates was chose and spread on another new 50MMA agar plate every 48h. The NaAC supplementation was increased **in a stepwise manner from 40 g L-1 to 100 g L-1 in a gradient of 20 g L-1 to achieve high-NaAC tolerance.**
![](https://static.igem.wiki/teams/5226/engineering-img/1-2.webp)

**Test:**
We primarily assess the growth of the strain based on its **cell morphology** observed on the agar plate. **A monoclonal strain that appears whiter, fuller, and larger indicates better growth.** Due to **a marked decline in the strain's growth at a concentration of 100 g/L sodium acetate**, we ultimately chose *Halomonas* TD cultivated on a 50 MMA agar plate containing 80 g/L sodium acetate for whole genome sequencing, resulting in the selection of strain TD80.

**Learn:**
Based on the strain's growth performance, we ultimately selected TD80 as the most promising candidate for utilizing sodium acetate, with the intention of employing it in fermentation production. Consequently, **further exploration is needed to determine the optimal concentration of sodium acetate to use during fermentation.**

#### Second cycle: Optimization of fermentation conditions

**Design:**
To prepare TD80 for fermentation production, we have decided to first **explore the fermentation conditions** to enhance the outcomes of our subsequent experiments.

**Build:**
**We utilized sodium acetate as the sole carbon source and established concentration gradients of 20, 25, 30, 35, and 40 g/L** to investigate the optimal sodium acetate concentration for TD80 fermentation.

**Test:**
After 48 hours of cultivation in shake flasks, we **measured the dry weight and analyzed the PHB content using gas chromatography.** The specific procedures are outlined in the protocol.

**Learn:**
Although the OD600 increases with higher sodium acetate concentrations, **the PHB conversion rate peaks at a concentration of 35 g/L.** Therefore, we ultimately selected 35 g/L of sodium acetate as the carbon source for TD80 fermentation.
![](https://static.igem.wiki/teams/5226/engineering-img/1280x1280.webp)

## Introduction of formate assimilation pathway

#### ·First cycle: Investigating the metabolism and tolerance of TD80 to sodium formate

**Design:**
Previously, the formate assimilation pathway has typically been studied in Escherichia coli[1][2], Cupriavidus necator[3], Vibrio natriegens[4] and other strains[5]. However, no relevant studies have been conducted on *Halomonas* TD. Therefore, before constructing plasmids for formate assimilation, we firstly **assessed the metabolism and tolerance of TD80 to sodium formate.**

**Build:**
We used sodium formate as **the sole carbon source**, and set the **concentration gradients** of 0, 5, 10, 15, 20, 25, 30, 35, and 40 g/L to explore the **metabolism** of sodium formate by TD80. Simultaneously, we introduced sodium formate with the above concentration gradient while maintaining a carbon source level of **35g/L sodium acetate** to evaluate the **tolerance** of TD80 to sodium formate.

**Test:**
We inoculated monoclonal colonies of TD80 into a deep well plate system containing 1ml of 50MM medium, supplemented with the carbon sources as described above. After 24 hours of cultivation in a shaker at 37 ℃ and 1000rpm, we measured the **OD600** of the bacterial solution using spectrophotometer. The **remaining concentrations** of sodium formate and sodium acetate were then analyzed using a liquid chromatograph.
![](https://static.igem.wiki/teams/5226/engineering-img/2-3.webp)
![](https://static.igem.wiki/teams/5226/engineering-img/3-3.webp)

**Learn:**
·Due to the significant error associated with the deep well plate, a 150ml shake flask was used as the container for the subsequent experiment.

·Based on the analysis of the data from the sodium formate tolerance experiment, we observed that the decrease trend of OD600 was not significant when the concentration of sodium formate was ≤ 35g/L. Therefore, for the fermentation experiment, we tentatively **set the concentration of sodium formate at 35g/L.**

[1] Yishai O, Bouzon M, Doring V, et al. In vivo assimilation of one-carbon via a synthetic reductive glycine pathway in Escherichia coli[J]. ACS synthetic biology, 2018, 7(9): 2023-2028.

[2] Kim S, Lindner S N, Aslan S, et al. Growth of E. coli on formate and methanol via the reductive glycine pathway[J]. Nature chemical biology, 2020, 16(5): 538-545.

[3] Claassens N J, Bordanaba-Florit G, Cotton C A R, et al. Replacing the Calvin cycle with the reductive glycine pathway in Cupriavidus necator[J]. Metabolic Engineering, 2020, 62: 30-41.

[4] Tian J, Deng W, Zhang Z, et al. Discovery and remodeling of Vibrio natriegens as a microbial platform for efficient formic acid biorefinery[J]. Nature Communications, 2023, 14(1): 7758.

[5] Turlin J, Dronsella B, De Maria A, et al. Integrated rational and evolutionary engineering of genome-reduced Pseudomonas putida strains promotes synthetic formate assimilation[J]. Metabolic Engineering, 2022, 74: 191-205.

#### ·Second cycle: Build and test pSEVA321-Mmp1-C1M

##### Iteration 1: Build pSEVA321-Mmp1-C1M

**Design:**
**The most critical reaction module** in the formate assimilation pathway is the **tetrahydrofolate (THF) cycle**, which is absent in TD80. C2M and C3M are endogenous to TD, but their expression intensity is relatively low based on existing laboratory data. To optimize the expression intensity of C1M for *Halomonas* TD, we have selected the inducible promoter Mmp1 to construct the C1 module plasmids for both AM1 and Vib.

**Build:**
We used the pSEVA321 scaffold with **Mmp1 inducible promoter** and chloramphenicol resistance gene.

The DNA sequences of AM1-C1M, ftfl, fch, and mtdA were synthesized by BGI Genomics Co.,Ltd. (Shenzhen,China);

The DNA sequences of Vib-C1M, ftl, and folD are derived from the genome of Vibrio. Natriegens.

The primers were synthesized by Songong Co., Ltd (Shanghai,China), accompanied by homologous arms, for assembly using Gibson assembly kit after PCR amplification.

Assembly products were subsequently converted into competent cells of Escherichia coli S17-1.
![](https://static.igem.wiki/teams/5226/engineering-img/4.webp)

**Test:**
Colonies were screened for desired constructs using colony PCR. Plasmids from positive clones were purified and digested with restriction endonucleases to preliminarily verify the success of cloning. Subsequently, Sanger sequencing (Songong Co., Ltd.(Shang hai, China)) was performed on the cut fragments to exclude the possibility of gene mutations.

**Learn:**
By comparing the sequencing results, it was found that there was no mutation in the genes. Therefore, the successfully constructed plasmid can be transferred into TD80 for fermentation validation.

##### Iteration 2: Fermentation testing of pSEVA321-Mmp1-C1M

**Design:**
Due to the synthesis of the AM1 genes by the company, pSEVA321-Vib-C1M was prioritized and underwent fermentation validation. we decided to **use 35g/L sodium formate as the sole carbon source** and make adjustments based on the results. Additionally, initial **IPTG induction concentration gradients** of 0, 2, 5, 10, and 20mg/L were set to activate the Mmp1 inducible promoter. Based on resistance gene screening, 25 μ g mL-1 chloramphenicol was added to the fermentation broth.

**Build:**
![](https://static.igem.wiki/teams/5226/engineering-img/5.webp)
Please refer to the protocol for details.

**Test:**
Before fermentation, it was observed that the solution in the shake flask became turbid, raising concerns that this turbidity might affect the OD600 measurements. As a result, only the remaining concentration of sodium formate was measured in the end.
![](https://static.igem.wiki/teams/5226/engineering-img/4-3.webp)

**Learn:**
Through a series of gradient turbidity tests and initial assessments of sodium formate tolerance, we adjusted the concentration of sodium formate during fermentation to **15g/L.** Additonally, based on the data obtained from the liquid chromatography, it was determined that it was too early to measure the remaining concentration of sodium formate. Therefore, for the time being, **only OD600 measurement** will be carried out to reflect the metabolism of sodium formate by cells.

![](https://static.igem.wiki/teams/5226/engineering-img/7.webp)
After the successful construction of pSEVA321-AM1-C1M, we conducted comparative fermentation using adjusted fermentation conditions. The experimental results showed that the introduction of AM1-C1M increased the growth of TD80 by **67.33%** under the culture conditions with sodium formate as the sole carbon source, while the introduction of Vib-C1M increased it by **59.67%**

![](https://static.igem.wiki/teams/5226/engineering-img/5-3.webp)
[6] Fu XZ, Tan D, Aibaidula G, Wu Q, Chen JC, Chen GQ. Development of *Halomonas* TD01 as a host for open production of chemicals. Metab Eng 23, 78-91 (2014).

#### ·Third cycle: Optimize C1M to eliminate the need for inducer

**Design:**
Given that the Mmp1 promoter requires IPTG for induction, which can be costly, and considering the challenges posed by concentration gradients in large industrial fermenters—where it is difficult to ensure uniform IPTG distribution to all bacteria—we propose converting the inducible promoter into a constitutive promoter.

Previous fermentation data indicated **no clear trend in stepwise changes under IPTG concentration gradients.** In fact, even at an IPTG concentration of 0, the expression intensity was higher. Therefore, we have decided to **replace the Mmp1 inducible promoter with two constitutive promoters, porin194 and porin281, which exhibit weaker expression intensities.**

After discussing this with our teacher, we considered that RBS2000 had previously been used as the ribosome binding site for C1M, and it yielded the highest expression intensity among the series of RBSs. Therefore, it would not be meaningful to focus solely on weakening the promoter at this stage. Instead, while adjusting the promoter strength, we decided to **change the RBS to the moderately intense B0064 for optimization.** Additionally, we **divided the C1M gene into two segments for tuning**, hoping to achieve varied results.

Based on our own insights and our teacher's advice, we ultimately decided to construct tuning plasmids with RBS B0064 and promoter tuning as the experimental group, while creating control plasmids that only incorporate weak promoter tuning.

**Build:**
For the construction of tuning plasmids, each group of genes, having their own promoters, was assembled through **a two-step reaction process**. Firstly, we constructed individual plasmids for each group of genes separately, and then combined the two groups of genes into a complete C1M.

In the first reaction, we utilized a pSEVA321 backbone containing the corresponding porin constitutive promoter, a chloramphenicol resistance gene, and RBS2000, along with a single C1M gene as the target fragment. A set of primers was designed at RBS2000, and due to the only difference in the base groups between B0064 and RBS2000 sequences, the RBS was directly modified during primer design. We then performed Gibson assembly to obtain pSEVA321-porin281/194-B0064-AM1-ftfl, AM1-fch-mtdA, Vib-ftl, and Vib-folD.

In the second reaction, two sets of tuning plasmids corresponding to the complete C1M were spliced together. Considering that the length difference between the assembled two fragments would reduce the success rate, we flexibly adapted and disconnected the pSEVA321 skeleton from the chloramphenicol resistance gene, with the former group of genes carrying the initial half of the skeleton and the latter group of genes carrying the second half of the skeleton. If the transformed strain can grow monoclonal antibodies on LB plates containing chloramphenicol, it indicates that the connection at the skeleton fracture is correct. During sequencing, only the genes at the interface of the two groups of genes need to be tested for mutations.

For the construction of plasmids with only weak promoters, the pSEVA321 backbone with dblTherm terminator, corresponding porphyrin constitutive promoter, and chloramphenicol resistance gene was used. The RBS2000-C1M sequence of the previously constructed pSEVA321-C1M plasmid served as the target fragment for Gibson assembly.

we conducted colony PCR to screen the colonies for the desired construct. Plasmids from positive clones were purified and digested with restriction endonucleases to preliminarily verify the success of cloning. Subsequently, Sanger sequencing (Songong Co., Ltd.(Shang hai, China)) was performed on the cut fragments to exclude the possibility of gene mutations.
![](https://static.igem.wiki/teams/5226/engineering-img/20.webp)

**Test:**
Using the same seed culture preparation scheme as in the second cycle, the secondary seed culture medium was inoculated into 20ml of 50MM-F medium at a volume of 5%, which contained 15g/L sodium formate and 25 μ g mL-1 chloramphenicol. The medium was incubated at 37 ℃ and 220rpm in an incubator for 48 hours.

After incubation, dilute the bacterial solution 5 times and measure the OD600 using a spectrophotometer. Dilute the bacterial solution 10 times and measure the remaining concentration of sodium formate using liquid chromatography.
![](https://static.igem.wiki/teams/5226/engineering-img/10-3.webp)

**Learn:**
Analysis of the data revealed that the **B0064-porin281 Vib ftl-porin194-Vib folD** group had the **lowest residual sodium formate concentration**, which was **15.3% lower** than that of the wild TD80 group. However, regarding **OD600 measurements, B0064-porin194-AM1 ftfl-porin281-AM1 fch mtdA** exhibited the best growth, showing a **66.5% increase** compared to wild TD80.

Considering liquid chromatography as a **quantitative analysis**, the amount of formate assimilated by TD80 was accurately determined. Furthermore, the difference in OD600 between the two groups was not significant. Finally, the combination of **B0064-porin281-Vib ftl-porin194-Vib folD** was selected as the most effective.

#### ·Fourth cycle: dual carbon source fermentation

**Design:**
Given our hope that *Halomonas* TD can utilize CO2 electrochemical derivatives instead of formate as their sole carbon source for growth, we initially discovered that the THF system effectively supports *Halomonas* TD using formate as a single carbon source. Building on this, we began to **explore whether the THF system could facilitate dual carbon source fermentation by primarily using acetate, while allowing TD80 to absorb and utilize formate.**

**Build:**
(1) Experimental Group
Recombinant TD80 with pSEVA321-B0064-porin281 Vib ftl-porin194 Vib folD. **Set sodium acetate concentration gradients at 0, 2, 4, 6, and 8 g/L, while maintaining a fixed sodium formate concentration of 5 g/L.** Monitor whether sodium formate absorption increases with rising sodium acetate concentrations.

(2) Control Group:
1. Wild-type TD80 without any carbon source, using only 50 mM as the cultural medium to eliminate the influence of other factors on TD80 growth.

2. Wild-type TD80 utilizing 5 g/L sodium formate as the sole carbon source, without the introduction of any plasmids.

3. Wild-type TD80 using 8 g/L sodium acetate as the sole carbon source, without the introduction of any plasmids.

4. Wild-type TD80 containing both 5 g/L sodium formate and 8 g/L sodium acetate, without the introduction of any plasmids.

**Test:**
To ensure the accuracy of the spectrophotometer, we diluted the bacterial solution to OD600 between 0.2 and 0.8 for measurement. After diluting the bacterial solution 10 times, measure the remaining concentrations of sodium formate and sodium acetate using liquid chromatography.

**Learn:**
The experimental results indicated that **as the concentration of sodium acetate increased, the absorption and utilization of sodium formate by TD80 also rose.** At a sodium acetate concentration of 8 g/L, the group supplemented with 5 g/L sodium formate **exhibited a 15% increase in growth** compared to the group without added sodium formate. At this stage, following the introduction of the formate assimilation pathway, *Halomonas* TD can **utilize the majority of CO2 electrochemical derivatives (CDE) for growth,** resulting in the production of easily recyclable, non-toxic bicarbonate in low concentrations.
![](https://static.igem.wiki/teams/5226/engineering-img/111.webp)

# ![flower](https://static.igem.wiki/teams/5226/general/before-h1-2.png)T-switch

#### ·First cycle: First try of exploration of useable thermosensitive bio-switch in *Halomonas* TD

**Design:**
After comparing the genome sequence of *Halomonas* TD with the genome sequence of E. coli, we found that the homology of the two is as high as 86.84%.Thus,we planned to put the thermosensitive bio-switch that is applicable to E. coli [1] in the *Halomonas* TD and test whether it could be used.

**Build:**
According to the literature[1],we constructed two plasmids with different copies:pSEVA321-prm-ci857-pr-sfgfp and pSEVA341-prm-ci857-pr-sfgfp.

**Test:**
Because *Halomonas* TD is easy to lose plasmids, we converted plasmid into E. coli s17-1 and transferred plasmids to *Halomonas* TD by junction and then characterisd it.However, the results was depressing——**none of them showed well performance.**
![](https://static.igem.wiki/teams/5226/engineering-img/8-3.webp)

**Learn:**
By analyzing the result, we speculated that the number of copies of the regulatory protein CI857 and the reported gene sfgfp in the thermalsensitive bio-switch shouldn't be expressed under the same number of copies .

#### ·Second cycle: Second try of exploration of useable thermosensitive bio-switch in *Halomonas* TD

**Design:**
we planned to separate the expression of ci857 from sfgfp and conducted it on plasmids with different copies in this cycle.We planned to try two different combinations of the plasmids: pSEVA321-prm-ci857+pSEVA341-pr-sfgfp and pSEVA341-prm-ci857+pSEVA321-pr-sfgfp.
![](https://static.igem.wiki/teams/5226/engineering-img/12.png)

**Build:**
As the first cycle, we used E.coli s17-1 to carry the plasmids and then expressed them in *Halomonas* TD.

**Test:**
Because sometimes it is difficult for two plasmids to join the strain at the same time, thus in each combination, we tried to join these two plasmids all at once and joined them one by one, and saw which one could succeed.**Surprisingly,the combination of 321-prm-ci857+341-pr-sfgfp performed great in controlling the expression of sfgfp.**

**Learn:**
It is an interesting phenomenon that our thermalsensitive bio-switch could only give full play to its role when two key genes are expressed on two certain plasmids.We thought the reason might be that when sfgfp was expressed on pSEVA321,its copies couldn't satifiy the expression of it. However, the copies of pSEVA321 was just enough to the expression of ci857.

![](https://static.igem.wiki/teams/5226/engineering-img/9-2.webp)

#### ·Third cycle: CI857 regulatory protein mutation

**Design:**
After we successfully built the basic thermalsensitive bio-switch for *Halomonas* TD, we wanted more.Thus, we decided to **mutate the regulatory protein** to see if we could get a version of CI857 with lower leakage,which could make the thermalsensitive bio-switch more rigorous.

**Build:**
We constructed the ci857 gene mutation fragment through a low-mutation rate random mutation kit. After measuring the concentration of the gene fragment, Gibson connection was carried out, and then all colonies on the transformation plate were characterised（the method could refer to previous cycle).

**Test:**
Like the second cycle, we joined this two plasmids into *Halomonas* TD and see how they worked by OD600 and FI under low and high temperature.By analyzing the result, we selected several well-performed mutants for future transfor-mation.

**Learn:**
We found several CI857 mutants with different fold changes, which meant their ability to express themselves in defferent temperatures were different. We could use this point **to adapt various genes' expressions.**

![](https://static.igem.wiki/teams/5226/engineering-img/30.png)

##### - Fourth cycle: TCI-42 regulatory protein mutation

**Design:**
After we successfully built the basic thermalsensitive bio-switch for Halomonas TD, we wanted more.Because Halomonas TD grew at 30°C in a mediocre manner, we sought out a narrower temperature region, the CI857 variant, TCI-42. And we decided to **mutate the regulatory protein** to see if we could get a version of TCI-42 with lower leakage, which could make the thermalsensitive bio-switch more rigorous.

**Build:**
Since TCI-42 is a mutant of CI857 with different characteristic. We tested it in Halomonas TD and constructed the TCI-42 gene mutation fragment through a low-mutation rate random mutation kit. After measuring the concentration of the gene fragment, Gibson connection was carried out, and then all colonies on the transformation plate were characterised（the method could refer to previous cycle).

**Test:**
Like the second cycle, we joined this two plasmids into Halomonas TD and see how they worked by OD600 and FI under low and high temperature.By analyzing the result, we selected several well-performed mutants for future transformation.

**Learn:**
We found several TCI-42 mutants with different fold changes, which meant their ability to express in low and high temperatures were different. We could use this point **to adapt various genes' expressions.**

![](https://static.igem.wiki/teams/5226/engineering-img/31.png)
![](https://static.igem.wiki/teams/5226/engineering-img/32.png)

##### ·Fifth cycle: promoter replacement of sfGFP and CI857

**Design:**
Because the expression intensity of the promoter pRM is relatively weak, we put forward a conjecture: if the promoter with strong expression intensity is used to express ci857, will the leakage under 30℃ be further reduced? Parts J23100 through J23119 (Designed by iGEM06\_Berkeley) are a family of constitutive promoter parts isolated from a small combinatorial library. Based on this conjecture, we chose the J23 series promoter for experimental verification.

**Build:**
The purpose of replacing the promoter was achieved by building the J23 series promoter (J23100-J23119) on the primer, and then connecting the fragments through the T4 connection. However, in the end, we only successfully built plasmids carrying promoter J23100 and J23110.

**Test:**
We tested this two plasmids acting with p341-pR-sfgfp in *Halomonas* TD and saw how they worked by OD600 and FI under 30 and 37 ℃.By analyzing the result, we surprisingly found that p321-j23110-ci857 showed great potential ——**lower leakage** and expression under 37℃ similar to plasmid with pRM as the promoter, which means **higher fold change.**

**Learn:**
We have confirmed that the strategy of improving the expression of ci857 to reduce leakage by replacing the promoter with a stronger expression intensity is feasible.

![](https://static.igem.wiki/teams/5226/engineering-img/33.png)
![](https://static.igem.wiki/teams/5226/engineering-img/34.png)

# ![flower](https://static.igem.wiki/teams/5226/general/before-h1-2.png)Products

## P34HB

#### First cycle: Introduction of P34HB synthesis pathway

The existing methods for large-scale production of P34HB primarily rely on microbial fermentation. A key limiting factor in this process is **the molar ratio of 4HB**. Increasing the 4HB molar ratio can lead to a decrease in the melting temperature and apparent fusion heat of the copolymer, as well as an improvement in the polymer's deformation resistance. Therefore, enhancing the molar ratio of 4HB is crucial for the modification of P34HB.

##### Iteration 1: Build 341-4hbd sucD ogdA-orfZ

**Design:**
Prior to embarking on this project, our laboratory had already conducted research on the production of P34HB. It was found that the expression of the 4hbd-sucD-ogdA-orfZ gene cluster could increase the molar ratio of 4HB. Following fermentation using Mmp1 inducible promoter, **the porin194 constitutive promoter was considered more suitable** based on the concentration gradient induction trend observed with IPTG.

Since two plasmids, pSEVA321 and pSEVA341, are commonly used in the laboratory, the gene cluster has only been previously expressed through the pSEVA321 plasmid. Our intention is to introduce the porin194-4hbd-sucD-ogdA-porin194-orfZ gene cluster into TD80 to synthesize P34HB, **utilizing both the pSEVA341 and pSEVA321 plasmids,** which allow us to evaluate which plasmid yields better results.

**Build:**
We obtained pSEVA321-porin194-4hbd-sucD-ogdA-porin194-orfZ directly from the laboratory. For pSEVA341-porin194-4hbd-sucD-ogdA-porin194-orfZ, we utilized the pSEVA341 backbone, which includes spectinomycin resistance gene and kanamycin resistance gene. The 4hbd-sucD-ogd-orfZ gene cluster was sourced from the existing pSEVA321 plasmid.

The primers were synthesized by Songong Co., Ltd (Shanghai,China), accompanied by homologous arms, for assembly using Gibson assembly kit after PCR amplification.

Assembly products were subsequently converted into competent cells of Escherichia coli S17-1.

**Test:**
We used colony PCR for preliminary screening, followed by Sanger sequencing to rule out the possibility of gene mutations. Please refer to the protocol for more details.

**Learn:**
By comparing the sequencing results, it was found that there was no mutation in the genes. Therefore, the successfully constructed plasmid can be transferred into TD80 for fermentation validation.

##### Iteration 2: Fermentation production of P34HB

**Design:**
Due to the incomplete optimization of formate assimilation, P34HB production initially was carried out in TD80 first. Based on the results of acetate domestication, we have decided to **use 35g/L sodium acetate as the sole carbon source.**

**Build:**
![](https://static.igem.wiki/teams/5226/engineering-img/13.webp)

**Test:**
After 48 hours of cultivation, we measured the dry weight of the strain and used gas chromatography to measure the 4HB molar ratio and PHA production. Please refer to the protocol for details.
![](https://static.igem.wiki/teams/5226/engineering-img/10-2.webp)

**Learn:**
The experimental results indicated that **the 4HB molar ratio** achieved by introducing the pSEVA321 was higher than that of the pSEVA341. However, the **dry weight** decreased somewhat compared to the wild TD80.

#### Second cycle: Change screening pressure

To **stabilize cell dry weight while increasing the 4HB molar ratio,** we investigated the effect of the pSEVA321 on cell dry weight. We hypothesized that the addition of chloramphenicol during the fermentation process impacted cell growth. Consequently, we decided to adjust the screening pressure and knock out cysNC, a gene encoding a key enzyme in the sulfate assimilation pathway, in *Halomonas* TD to **block the supply of sulfur sources for methionine synthesis.** Simultaneously, we incorporated the cysNC gene into the pSEVA321 backbone to screen for strains that had been successfully transformed with the 321-porin194-4hbd-sucD-ogdA-porin194-orfZ plasmid.

##### Iteration 1: Constructing knockout plasmids

**Design:**
We used CRISPR/Cas9 for gene knockout. Due to the challenges of gene editing in *Halomonas* TD, we employed two methods to block the expression of cysNC. The first method involved **knocking out all codons from the start codon (ATG) to the stop codon.** The second approach entailed **knocking out the core region from its full-length sequence.** We designed **two sets of gRNAs** targeting the specific regions for each knockout method. Both methods were performed simultaneously, and successful knockouts were selected for the subsequent fermentation step.

**Build:**
We first designed primers to amplify the 1000 bp homologous arms on either side of the core region of the cysNC gene, and the vector backbone. Then, through the Gibson assembly process, we linked the left and right homologous arms with the vector.

After successful sequencing, we designed two pairs of primers to modify the guide RNA in each plasmid. The entire plasmid was divided into two parts at the kanamycin resistance gene to use a medium containing kanamycin for screening bacteria that have successfully taken up the ligated plasmids.

**Test:**
We used colony PCR for preliminary screening, followed by Sanger sequencing to rule out the possibility of gene mutations. Please refer to the protocol for more details.

**Learn:**
By comparing the sequencing results, it was found that there was no mutation in the gene. Therefore, the successfully constructed plasmid can be used to knock out cysNC or its core region in the TD80 genome.

##### Iteration 2: Knock out cysNC

**Design:**
After constructing pSEVA341-gRNA, we used CRISPR/Cas9 to knock out cysNC or its core region in TD80.

**Build:**
Plasmids were transferred from E. coli S17-1 to H. bluephagenesis TD80 using an optimized conjugation transformation protocol as follows: E. coli S17-1 as the donor cell which harbors the plasmid was cultured in LB medium in the presence of appropriate antibiotic(s). H. bluephagenesis TD80 0r recombinant H. bluephagenesis TD80 were the recipient cells, and were cultured in 60LB. Select monoclonal antibodies and culture them overnight in 5 mL of the appropriate medium. Next, take 20 µL of both the recipient bacterial solution and the donor bacterial solution, and mix them on a 20 LB plate. Incubate the mixture overnight at 37 °C. Finally, resulting bacterial lawn was re-suspended in 60LB medium and spread on a 60LB agar plate with appropriate antibiotics, followed by incubation at 37 °C for 24–48 h to select for transconjugants.
![](https://static.igem.wiki/teams/5226/engineering-img/15.webp)

Initially, S17-1 (pSEVA321-Cas9) was conjugated with TD80 and subsequently cultured on a **60AC plate** containing ampicillin and chloramphenicol. After achieving monoclonal growth, we obtained **TD80 (Cas9)** by streaking on the 60AC plate.

Following the acquisition of TD80 (Cas9), it was conjugated with **S17-1 (pSEVA341 gRNA)** and cultured on a **60CSM plate** containing chloramphenicol, spectinomycin, and methionine. Once monoclonal antibodies grow, we can verify the successful knockout of cysNC or its core region.

**Test:**
After growing monoclonal colonies on a 60CSM plate containing chloramphenicol, spectinomycin, and methionine, we designed a pair of **universal primers** at 50-100bp around the homologous arm of the genome, along with a **specific primer** located in the middle of the target knockout gene.
![](https://static.igem.wiki/teams/5226/engineering-img/16.webp)

First, we performed colony PCR using the universal primers and sampled the bacteria on a 60LB plate. If the knockout was successful, we expected to see **a band around 4500 bp**; if it failed, the band would be around 2200 bp. On the following day, we conducted **a secondary validation** on the successfully knocked-out strains using the specific primers. If the knockout was successful, **no bands would be present**; if it failed, a band approximately 2000 bp in length would appear.

After successful double validation, in order to facilitate subsequent gene editing and plasmid transfer of the strain, we used a deep well plate to eliminate resistance in the strain at a temperature of 42 °C. The deep well plate consisted of 1 ml of 60LB and **0.2 g/L methionine**, and the cultures were incubated for 24 hours.

Starting from the third generation, we verified whether resistance had been removed. Since pSEVA321-Cas9 is easily eliminated, our primary focus was to verify whether pSEVA341-gRNA-cysNC had been removed. We took 5 μL of the bacterial solution and transferred it to a **60ASM plate** containing spectinomycin and methionine, followed by incubation at 37 °C for 12 hours. If no colonies grew, this indicated that resistance had been successfully removed.

The next generation was then streaked to select monoclonal colonies from the 60M plate containing methionine for double validation. If successful, the bacteria could be preserved; if not, it would indicate that the bacteria on the sample plate had reverted to the wild type. In such a case, we needed to re-verify whether the bacteria on the sample plate had returned to wild type. If they had, the coated plate should be reconnected; if there was no response, we would begin the de-antibody passage and repeat the above steps until success was achieved.

**Learn:**
After numerous attempts, t we successfully achieved double validation and the removal of the antibody. The sequencing outcomes revealed that the core region of the cysNC had been successfully knocked out. We can now proceed to the next step of the experiment, which involves constructing cysNC on pSEVA321 as a new selection pressure.

##### Iteration 3: Build pSEVA321 (C+cysNC) - porin194-4hbd-sucD-ogdA-porin194-orfZ

**Design:**
After blocking the expression of cysNC in TD80, the strain is unable to synthesize methionine, making it difficult to grow in methionine-deficient media. We aim for only the recombinant TD80 containing the porin194-4hbd-sucD-ogdA-porin194-orfZ gene cluster to thrive in 50 mM medium without the addition of antibiotics. To achieve this, we incorporated the cysNC gene into the pSEVA321 backbone, allowing TD80 transformed with the plasmid to synthesize methionine and grow in 50 mM medium lacking methionine.

**Build:**
First, a pair of primers was designed to obtain the RBS for cysNC from the genome using PCR. Next, we designed two pairs of primers to divide pSEVA321-porin194-4hbd-sucD-ogdA-porin194-orfZ into two segments, located after the chloramphenicol resistance gene and the 4-hbd gene, respectively. While designing the primers, we included homologous arms to facilitate the connection of these three segments through Gibson assembly, resulting in the formation of a new plasmid: pSEVA321 (C+cysNC) - porin194-4hbd-sucD-ogdA-porin194-orfZ.

**Test:**
We used colony PCR for preliminary screening, followed by Sanger sequencing to rule out the possibility of gene mutations. Please refer to the protocol for more details.

**Learn:**
By comparing the sequencing results, it was found that there was no mutation in the genes. Therefore, the successfully constructed plasmid can be transferred into TD80-△cysNC for fermentation validation.

##### Iteration 4: test pSEVA321 (C+cysNC) - porin194-4hbd-sucD-ogdA-porin194-orfZ

**Design:**
After successfully knocking out the key region of the cysNC gene and constructing pSEVA321 (C+cysNC)-porin194-4hbd-sucD-ogdA-porin194-orfZ, we initiated verification to determine whether altering the screening pressure could enhance the 4HB molar ratio.

**Build:**
We have designed the following experimental groups:

##### 1.TD80（△cysNC-key）&321(C + cysNC) -194-4hbd-sucD-ogd-194-orfZ

This was the key experimental group aimed at determining whether optimal screening pressure was beneficial to the growth of TD80 compared to Group 2.

##### 2.TD80 & 321(C) -194-4hbd-sucD-ogd-194-orfZ

As the control group for Group 1, it was essential to compare whether optimizing the screening pressure had a beneficial effect on the growth of TD80.

##### 1. TD80（△cysNC-key）

To determine **whether the knockout of cysNC-key impairs TD80's ability to synthesize methionine.**

##### 2.TD80（△cysNC-key）&0.2g/L Met

If Group 3 demonstrated that TD80 (△cysNC-key) could not synthesize methionine, **the effect of cysNC supplementation in the form of pSEVA321 on the growth of strains** was then observed in this group.

##### 5.TD80

Examine the ability of wild strains to produce P34HB.

**Test:**

**Learn:**
By plotting the growth curves, it is evident that the growth of TD80 (△cysNC-key) is comparable to that of TD80 and TD80 (△cysNC-key) in the presence of 0.2 g/L Met. Consequently, cysNC is not an essential gene in the methionine synthesis pathway and cannot be used as a selective pressure to ensure continuous expression of the gene cluster in TD80.
![](https://static.igem.wiki/teams/5226/engineering-img/28-1.png)

After gas phase analysis, the molar ratio of P34HB produced by TD80 (△cysNC-key) & 321(C+cysNC)-194-4hbd-sucD-ogd-194-orfZ was significantly lower than that of TD80 & 321(C)-194-4hbd-sucD-ogd-194-orfZ. This suggests that some TD80 cells lost the pSEVA321 plasmid during fermentation and ceased to produce P34HB.
![](https://static.igem.wiki/teams/5226/engineering-img/29.png)

The dry weight results indicated that the growth of recombinant TD80 increased even without the addition of antibiotics. Therefore, identifying a new essential gene may be a viable strategy to enhance the 4HB mol% of P34HB, and this will be a focus of our future efforts.

## Amino acid derivatives

### Tyrian Purple

#### ·First cycle: Introduce the tyrian purple synthesis pathway in *Halomonas* TD

**Design:**

**Build:**
There were four genes that we needed to introduce to *Halomonas* TD:stth,fre,tnaA and fmo.Their specific information could be seen in our part page. For stth and fmo,we obtained them from the genome of E.coli 1655.As for fre and tnaA,we entrusted the company to help synthesise the corresponding gene fragments.Then, we built two plasmids separately carrying these four genes according to the synthesis pathway:pSEVA341-fre-stth and pSEVA321-tnaA-fmo.At last, we transformed this two plasmids into a same strain *Halomonas* ΔAFTD,which knocked out pheA and pykF. It was proved that the knock-out of these two genes could improve production of tryptophan, the precursor of tyrian purple[2][3].We obtained the strain from our laboratory.

**Test:**
We used the constructed strain for 48-hour shake flask fermentation. After the fermentation, the sample was processed and they all seemed **depressingly blue**, which means there were a large amount of by-product indigo. Because the standard sample of tyrian purple would not arrive for some time, we decided to do some other experiments to see whether the pathway in *Halomonas* ΔAFTD could work.
![](https://static.igem.wiki/teams/5226/engineering-img/6-3.webp)

**Learn:**
Before we could know that whether the strain could produce Tyrian purple, first we should make sure that there were enough 6-Br-Trp provided for follow-up reaction. Thus, before the standard sample of Tyrian purple arrived, we should do another reasearch.

reference:

[1]Lee, J., Kim, J., Song, J. E., Song, W.-S., Kim, E.-J., Kim, Y.-G., Jeong, H.-J., Kim, H. R., Choi, K.-Y., & Kim, B.-G. (2021). Production of Tyrian purple indigoid dye from tryptophan in Escherichia coli. Nature Chemical Biology, 17(1), 104–112.

[2]Guo L, Ding S, Liu Y, Gao C, Hu G, Song W, Liu J, Chen X, Liu L. Enhancing tryptophan production by balancing precursors in Escherichia coli. Biotechnol Bioeng. 2022 Mar;119(3):983-993. doi: 10.1002/bit.28019. Epub 2021 Dec 30. PMID: 34936092.

[3]Xiong B, Zhu Y, Tian D, Jiang S, Fan X, Ma Q, Wu H, Xie X. Flux redistribution of central carbon metabolism for efficient production of l-tryptophan in Escherichia coli. Biotechnol Bioeng. 2021 Mar;118(3):1393-1404. doi: 10.1002/bit.27665. Epub 2021 Jan 13. PMID: 33399214.

#### ·Second cycle: Find out whether the strain could produce enough substrate for follow-up reaction

**Design:**
We planned to transform the plasmid that is responsible for the production of 6-Br-Trp into the strain and use the strain to test if it could produce enough substrate for follow-up reaction.

**Build:**
We transformed pSEVA341-mmp1-fre-stth into *Halomonas* ΔAFTD.

**Test:**
We designed four experimental groups:IPTG=5,20(mg/L) and Trp=0.4,0.8(g/L).Each group set up three parallel samples and all of them were cultivated 48h.Samples were taken at 24h,36h and 48h.After the fermentation, we processed the samples and analyzed the product in it. The result showed that at 24h the amount of 6-Br-Trp reached its peak.

**Learn:**
Our experiment showed that the reaction of using tryptophan to produce 6-Br-Trp in *Halomonas* ΔAFTD is feasible, which greatly encourages us. In fact, the results of this experiment also provide a reference for the time to switch the temperature after the subsequent introduction of thermosensitive bio-switch.
![](https://static.igem.wiki/teams/5226/engineering-img/11-1.webp)

#### ·Third cycle: Introduce thermosensitive bio-switch into the synthesis of Tyrian purple

**Design:**
Since in the first cycle the fermentation result seemed depressingly blue, we kept searching information to get the answer. By reading papers we got an important information that Stth and TnaA were isoenzyme, which means they could use tryptophan as substrate to produce different products. When TnaA reacts with the substrate, a large number of indigo by-products are produced, and studies show that tryptophan has a higher preference for TnaA, which means more by-products and fewer target products.Thus, we need to separate this two enzymes to make sure enough substrate for Stth.It was at that time that we came up with the thought of introduce thermosensitive bio-switch into the synthesis of tyrian purple.When looking for relevant literature, we saw a newly published literature this year that also introduced thermalsensitive bio-switch in the production process of tyrian purple to separate the expression of Stth and TnaA enzymes, which finally achieved good results[1].It greatly encouraged us, because it meant that our ideas were feasible.

**Build:**
When constructing the T-switch, we found that the dynamic regulation performance of the switch was better when switching from low temperature to high temperature. Therefore, when applying to the production of Tyrian purple, we also planned to use the switching from low temperature to high temperature to realise the separate expression of Stth and TnaA. Therefore, we constructed two plasmids, pSEVA341-pr-mmp1-tnaA-fmo and pSEVA321-prm-porin58-fre-porin68-stth, and verified by fermentation.

**Test:**
We designed to test four strains as below:

| No. | Strain | Usage |
| --- | --- | --- |
| 1 | ΔAFTD+pSEVA341-PRM/pr-tnaA-fmo+pSEVA321-pRM-ci857-mmp1-fre-stth | It was used to check the yield of tyrian purple. |
| 2(Only for deepwell plate fermentation) | ΔAFTD+pSEVA341-PRM/pr-sfgfp+pSEVA321-pRM-ci857-mmp1-fre-stth | It was used to check the leakage of T-switch. |
| 3 | ΔAFTD+pSEVA341-PRM/pr-tnaA-fmo | As a control of the first strain to assess the yield of Tyrian purple |
| 4 | ΔAFTD+pSEVA321-pRM-ci857-mmp1-fre-stth | It was used to test the yield of 6-Br-Trp at different temperatures. |

Other variables were the amount of IPTG and tryptophan added.We set two gradients for each of the two variables:IPTG(mg/L)=5, 20 and Trp(g/L)=0.4,0.8.

At 16,24,32h, we took a batch of samples and switched the temperature to 37℃,in case to find a better timing to switch temperature through the yield of 6-Br-Trp.

Strain1, 3 and 4 was for shake flask fermentation to test the production of Tyrian purple.Strain2 was for deepwell plate fermentation to test the leakage of our T-switch in the synthesis of Tyrian purple as a control group.

**Learn:**

![](https://static.igem.wiki/teams/5226/eng/eng1.jpg)
![](https://static.igem.wiki/teams/5226/eng/eng2.jpg)

![](https://static.igem.wiki/teams/5226/eng/eng3.jpg)
![](https://static.igem.wiki/teams/5226/eng/eng4.jpg)
![](https://static.igem.wiki/teams/5226/eng/eng5.jpg)
![](https://static.igem.wiki/teams/5226/eng/eng6.jpg)

We tested samples taken from group 1 and group 3 as experimental group and control group. The result showed that the experimental group produced 12.31 mg/L and 25.49 mg/L of tyrian purple under the condition of switching to 37°C to continue culture after 36 hours of cultivation at 30°C and keeping in 30°C in the whole process respectively.

![](https://static.igem.wiki/teams/5226/engineering-img/38-1.png)

The ratio of Tyrian purple to indigo yield expressed under the same switching temperature and Trp,IPTG protocol is as follows.

| Growing time at 30℃ | Ratio |
| --- | --- |
| 48h | 5.10 |
| 32h | 2.46 |

![](https://static.igem.wiki/teams/5226/engineering-img/39-1.png)
![](https://static.igem.wiki/teams/5226/engineering-img/40-1.png)
![](https://static.igem.wiki/teams/5226/engineering-img/41-1.png)
![](https://static.igem.wiki/teams/5226/engineering-img/42-1.png)

At the same time, we tested the fluorescence intensity(FI) of group 2 to see the expression intensity of pR/pRM-tnaA-fmo in different timing. The result showed that most of the fluorescence leakage were controlled below 20, and a small number of anomalous data are believed to be caused by the poor uniformity of the shaker, which led to the lack of strict temperature control.Fermentation results from deep-well plates showed that the separation of Stth and TnaA expression using our T-switch was effective.

reference:

[1]Feifei Li, Que Chen, Huaxiang Deng, Shumei Ye, Ruidong Chen, Jay D. Keasling, Xiaozhou Luo,One-pot selective biosynthesis of Tyrian purple in Escherichia coli,Metabolic Engineering,Volume 81,2024,Pages 100-109.

[2]Athina Vasileiadou, Ioannis Karapanagiotis, Anastasia Zotou, Determination of Tyrian purple by high performance liquid chromatography with diode array detection,Journal of Chromatography A,Volume 1448,2016,Pages 67-72.

[3]Athina Vasileiadou, Ioannis Karapanagiotis, Anastasia Zotou,

Determination of Tyrian purple by high performance liquid chromatography with diode array detection,Journal of Chromatography A,Volume 1448,2016,Pages 67-72，

## Proteins:SOD and phaP

**Design:**

**Build:**

**Test:**
After adding IPTG for 12 hours of incubation, we measured the dry weight and OD600 of the strains and performed protein expression assays by SDS-PAGE.Please refer to the protocol for details.
![](https://static.igem.wiki/teams/5226/engineering-img/17.webp)

**Learn:**
From the fermentation result graph, it can be seen that the exogenous genes phaP and SOD genes can be expressed in TD80, and the amount of protein produced by glucose as carbon source is more than that produced by sodium acetate as carbon source. In addition, the expression of exogenous phaP and SOD genes in TD80 had relatively little effect on the growth of TD80 itself.

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