iGEM 2023 — Team jcvi-ucsd

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# Plasmid Design and Construction Notebook

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# Thermal Adaptive Laboratory Evolution Notebook

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# GC-MS Notebook

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# Design Cycle

![engineering-cycle](https://static.igem.wiki/teams/4921/wiki/banners/sponsor/engineering-cycle.png)

# Building the POAP Cycle

![research](https://static.igem.wiki/teams/4921/wiki/icons/research-2.png)

### Research

We began our project by first familiarizing ourselves with the climate crisis, carbon fixation pathways, and current initiatives in carbon sequestration. In this effort, we collectively read literature that cover the science behind climate change, natural carbon fixation pathways, synthetic carbon fixation pathways, and current efforts in carbon capture through synthetic biology [1].

![imagine](https://static.igem.wiki/teams/4921/wiki/icons/data-science-2.png)

### Imagine

From our literature search, we were introduced to the two main categories of carbon fixation - natural carbon fixation pathways and synthetic carbon fixation pathways [2]. Synthetic carbon fixation pathways are human designed pathways that are pieced together using characterized enzymes. Because of this, synthetic carbon fixation pathways are well-characterized and can have higher carbon fixation rates than natural pathways. In particular, one literature that we drew inspiration the most from is the development of the pyruvate carboxylase (PYC), oxaloacetate acetylhydrolase (OAH), acetate-CoA ligase (ACS), and pyruvate synthase (PFOR) (POAP) cycle which is the first reported in vitro minimized carbon fixation pathway [3]. The simplicity of the pathway, due to its minimized enzymatic reactions, and high reported carbon fixation rates makes it an enticing candidate for in vivo applications. When selecting the biological chassis, we had to consider the need for easy analysis of pathway impact on biological functions because in vivo implementation of minimized carbon fixation cycles is unexplored. It is important for us to identify how such a pathway can affect biological processes and in response, find ways to optimize pathway compatibility and activity. Combined with considerations on biocontainment, safety, and ease of engineering, we chose the minimal cell (JCVI-Syn3B) to be our biological platform. The minimal cell is both the world’s simplest and synthetic cell. This means the minimal cell is very well characterized and grows only in specific laboratory conditions [4]. Its genome also contains a cre-loxp site, known as the landing pad, that allows for efficient integration of genes into the genome. This prompted us to imagine a carbon fixation system that is afforded by the POAP cycle and the minimal cell.

![design](https://static.igem.wiki/teams/4921/wiki/icons/petri-dish.png)

### Design

After imagining a carbon fixation system, we were led to the design of MACS - A Minimal, Adapted Carbon Sequestration system. The minimal part of the system arises from the placement of a minimized carbon fixation cycle into a minimal cell. The adapted part comes from the adaptive laboratory evolution (ALE) experiments we performed on the minimal cell to increase compatibility with the POAP pathway. Our first design of MACS entailed the selection of optimal variants for the enzymes used in the POAP cycle. One challenge we identified was that the POAP cycle activity was reported at 50 degrees Celsius in vitro which is hostile to the growth of minimal cells [3]. The main reason why the POAP cycle operates at this high temperature is because the pyruvate:ferredoxin oxidoreductase (PFOR) enzyme requires high temperature to catalyze pyruvate synthesis. Our first design goal is then to screen for variants of PFOR enzyme that have lower operating temperatures for pyruvate synthesis. Using the Braunschweig Enzyme Database (BRENDA), we looked at all characterized versions of PFOR from different organisms and finalized on Desulfocurvibacter africanus PFOR which has reported pyruvate synthesis activities at 40 degrees Celsius [5]. Likewise, we did the same search for all enzymes involved in the POAP cycle, using operating temperatures around 40 degrees Celsius and pH around 5-7 as screening criterias, and finalized on the following variants of enzymes to build our POAP cycle. Despite using the africanus PFOR, we were still concerned with the conditions being hostile for the minimal cells. Thus, we initiated an adaptive laboratory evolution project aimed at evolving minimal cell thermal tolerance (TALE) which runs parallel to our plasmid design and implementation cycle.

To finalize our design, we had to attach appropriate Mycoplasma promoters to our genes. By using characterized expression levels of Mycoplasma promoters identified in a transcriptomics study, we selected promoters with intermediate strength. These promoters, which have been registered as basic parts, were attached to the genetic sequences of our genes, and these sequences were ordered from Twist Biosciences. Because some gene fragments are large, Twist Biosciences opted to synthesize our gene fragments, namely Acetyl-CoA synthetase (ACS) and PFOR, in fragments of 2 and 3 respectively. Additionally, primers designed to amplify our genes and perform crossover PCR were also ordered from Twist Biosciences. We designed our building step to constitute assembly of individual genes into distinct plasmids. This affords us the ability to screen for the activity and biological compatibility of individual genes before assembly into the full POAP pathway. Combination of all 4 genes from the very beginning may lead to difficult troubleshooting in the face of obstacles since we will be more uncertain of where and how things failed.

| Gene | **Organism** | Organism Type | Working Temp (°C) | Gene Length (nt) |
| --- | --- | --- | --- | --- |
| PFOR | **Desulfocurvibacter africanus** | Bacteria | 40 | 3699 |
| PYC | **Bacillus subtilis** | Bacteria | 37 | 3443 |
| OAH | **Aspergillus niger** | Fungus | ≤45 | 1026 |
| OAH | Moniliophthora perniciosa | Fungus | 30 | 993 |
| OAH | Dianthus caryophyllus | Plant | 20 | 1267 |
| ACS | Dunaliella tertiolecta | Algae | 37-45 | 2464 |
| ACS | **Pseudomonas putida** | Bacteria | 38 | 1962 |
| ACS | Bacillus subtilis | Bacteria | 40 | 6300 |

![build](https://static.igem.wiki/teams/4921/wiki/icons/chemistry.png)

### Build

Our building efforts began upon the arrival of our ordered genes and primers. We first performed PCR on our genes to both obtain larger quantities of our gene fragments and to prepare for E. Coli plasmid assembly. Our second step is to perform crossover PCR to combine the two fragments of ACS into one and the 3 fragments of PFOR into one. We then began to assemble our plasmids by combining the pCC1BAC EcoRV fixed HindIII Exp Vector backbone (provided by the Suzuki Lab) with our gene inserts using E. Coli plasmid assembly. Successful assemblies will supply transformed E. Coli cultures with resistance to chloramphenicol which is used as a selection marker.

![test](https://static.igem.wiki/teams/4921/wiki/icons/magnifying-glass.png)

### Test

Even though successful assemblies of our plasmid will lead to transformed E. Coli cultures growing on a plate containing an antibiotic, colonies should still be picked and have their plasmids mini-prepped and sequenced to confirm that our genes are unmutated and installed correctly. After mini-prepping all of our transformed E. Coli cultures and sending them off for sequencing by Plasmidsaurus, we confirmed that we have individual plasmids containing each component of the POAP cycle.

![learn](https://static.igem.wiki/teams/4921/wiki/icons/cancer.png)

### Learn

During our engineering cycle, we encountered multiple challenges in the combination of PFOR and ACS gene fragments to yield the final gene. In our troubleshooting, we tested different primer combinations and conditions for crossover PCR which eventually yielded us successful combinations. This learning experience gave us a better understanding on the principles of PCR, how certain primers may work better than others, and PCR conditions that can be fine tuned to gene concentration and size. In this process, we also learned the principles and protocol for E. Coli plasmid assembly which works similarly to Gibson Assembly. Lastly, our use of a copy control vector exposed us to a brand new field of plasmids which can have their rate of replication carefully controlled. Overall, we were able to improve on our initial protocol of crossover PCR and E. Coli plasmid assemblies and produce successfully assembled plasmids with desired gene inserts.

# Transformation of Minimal Cell

![design](https://static.igem.wiki/teams/4921/wiki/icons/petri-dish.png)

### Design

After obtaining assembled plasmids in E. Coli and confirming their fidelity, the very next step is to transform minimal cells with those plasmids. What’s particularly interesting is that the vector we are using to carry our genes of interest becomes a suicide plasmid once inside of the minimal cell. But because the plasmid contains a cre-loxp site, our gene of interest (along with an antibiotic resistance gene), can be integrated into the minimal cell genome which also contains a cre-loxp site known as the landing pad. This allows for the transfer of our gene of interest into the minimal cell, and we can use an antibiotic to select for the cells that underwent this transformation. In our design, we selected puromycin to be this selection marker. We also wanted a way to confirm that our gene is indeed integrated into the minimal cell. Thus, we designed primers that complements our gene and would produce PCR products from the minimal cell genome.

![build](https://static.igem.wiki/teams/4921/wiki/icons/chemistry.png)

### Build

We proceeded to mini-prep more plasmids from E. Coli cultures that contain sequenced plasmids. These plasmids were used in Mycoplasma transformation, and we plated our minimal cell cultures onto SP4 agar plates containing puromycin.

![test](https://static.igem.wiki/teams/4921/wiki/icons/magnifying-glass.png)

### Test

Although we encountered challenges in transformation of certain plasmids, namely ACS and PFOR in the beginning, we eventually got transformed minimal cell colonies for all plasmids growing on puromycin SP4 agar plates. The growth of these colonies on puromycin plates alludes to the successful integration of our gene of interest into the minimal cell genome, but we needed a more concrete test. Thus, we performed PCR on our minimal cell culture using appropriate primers and ran them across an agarose gel. Their expected band size gives us confidence that our genes of interest are indeed integrated into the minimal cell genome.

![learn](https://static.igem.wiki/teams/4921/wiki/icons/cancer.png)

### Learn

In this engineering cycle, we learned new techniques such as Mycoplasma transformation as well as troubleshooting Mycoplasma transformation to get all of genes of interest integrated into the minimal cell. We were also exposed to the concept of suicide plasmids and became more familiar with the efficient integration of DNA using the cre-loxp system. The most valuable lesson we learned is a rigorous approach to troubleshoot and test things - including the use of PCR to confirm the presence of our gene in addition to just finding colonies on an antibiotic plate. From these lessons, we were able to continually improve our protocols, eventually getting transformations that failed in the beginning to work in the end.

# GC-MS

![research](https://static.igem.wiki/teams/4921/wiki/icons/research-2.png)

### Research

Because the metabolites involved in the POAP cycle are mainly primary metabolites such as pyruvate and acetate, we wanted to develop an extraction and derivatization protocol that affords us untargeted metabolic profiling. Further, untargeted metabolic profiling also allows us to see the overall metabolic state of the minimal cell, and we can compare the profile of a transformed minimal cell with the untransformed minimal cell to observe the overall perturbations caused by one component of the cycle or by the overall cycle. With this goal in mind, we searched through literature to find reports of untargeted metabolite profiling. We found one literature in the Plant Journal that fitted our needs and adapted their procedure, with changes, for the minimal cell [6].

![design](https://static.igem.wiki/teams/4921/wiki/icons/petri-dish.png)

### Design

One thing we had to consider in our design for the extraction of minimal cell metabolites is that the minimal cells are fragile and can be ruptured easily. Because of this, we lowered the centrifugation speed to 2,800x RCF to ensure that cells can be pelleted but are not ruptured. Another thing we had to consider is the necessity of washing the cell pellets. SP4 media, the media that minimal cells grow in, is rich in cofactors, nutrients, and salts which can add lots of noise to a GC-MS profile. We must add extra steps of cell pellet washing to remove these contaminants which may add noise to the GC-MS analysis. In designing this wash step, we decided to use a TRIS-Saline buffer to control the pH and maintain osmosis so the cells do not rupture. This wash buffer was designed to be 20 mM TRIS and 200 mM NaCl tuned to a pH of 7.5. In the derivatization portion of the protocol, another design decision was made to replace the lyophilization of extracted metabolites by solvents to be speed-vacuumed. This is both to save time (lyophilization will take overnight whereas speed-vacuum can evaporate solvents in 2 hours) and allows us to treat 12 samples at the same time (speed-vacuum we had access to can treat 12 samples at a time). The protocol for minimal cell metabolite extraction and untargeted metabolite profiling by GC-MS can be found in our Experiments section in this wiki.

![test](https://static.igem.wiki/teams/4921/wiki/icons/magnifying-glass.png)

### Test

With our developed extraction and profiling protocol, we processed multiple transformed minimal cell samples and developed standards to locate the derivatized product in the GC-MS trace. In our first round of experiment, we encountered two challenges that allowed us to learn and refine our protocol. The first challenge was the addition of organic solvents into plastic eppendorf tubes and the extraction of metabolites in these tubes. The concern is that organic solvents may strip plastic particles which may lead to noise later in the GC-MS analysis that can cover up smaller metabolic signals. Another challenge we encountered is the need for optimization of the GC-MS method. Because we are performing an untargeted metabolite analysis, metabolites of different size and polarities are screened with the GC-MS, from compounds that elute very quickly like pyruvate to compounds that elute much slower like lipids and cholesterol. We needed a GC-MS method that lends coverage for both the fast eluting metabolites and the slow ones, while also making sure that no metabolites are eluted so slowly such that they appear in the trace of a new run (which was actually observed initially in which slow-eluting metabolites of an old run appeared in the early elution phases of a new sample run).

![learn](https://static.igem.wiki/teams/4921/wiki/icons/cancer.png)

### Learn

From our two challenges encountered during the testing phase, we learned more about what needs to be considered in the extraction of metabolites, derivatization, and screening. Surprisingly, the GC-MS profiles from our first experiment were very clean with no noise. Thus, it is a pleasant surprise that extraction in plastic eppendorf tubes does not introduce plastic contaminants. In the case that extraction in plastic tubes will lead to contaminants, we could attempt to extract in glass vials by moving the cell pellets with a pipette tip to a glass vial prior to lyophilization. We can also avoid the centrifugation step post-extraction and just be very careful in picking up only the solvent and pipette it over a small filter before collection. From our challenges with the GC-MS profiling, we changed the settings of the GC with the help of our secondary PI, Dr. Immo Burkhardt, to have a broad coverage of derivatized metabolites. We also added an ethyl-acetate wash step in between each sample run to ensure that no metabolites from the previous run is eluted in a new run.

# TALE

![research](https://static.igem.wiki/teams/4921/wiki/icons/research-2.png)

### Research

A primary identified problem with the minimal carbon fixating cell design was that the cell cannot withstand temperature extremes where PFOR operates in the correct direction. Research to overcome this issue began with looking at PFOR variants that could operate at lower temperatures. A solution appeared after consulting with synthetic biology experts.

On November 7, 2022, we consulted Dr. Clyde Hutchinson about our design bottleneck. Dr. Hutchinson suggested that we try to grow the minimal cell colonies at slowly increasing temperatures. The idea would be to select out the temperature resistant mutants through natural selection.

![imagine](https://static.igem.wiki/teams/4921/wiki/icons/data-science-2.png)

### Imagine

On November 11, 2022, we consulted with Dr. John Glass about Dr. Hutchinson’s idea. Dr. Glass suggested a serial passaging experiment where the current JCVI Syn3.0B cell is initially grown at 39oC. Using a thermocycler machine, we can use the heat gradient setting to find a thermal bottleneck temperature.

![design](https://static.igem.wiki/teams/4921/wiki/icons/petri-dish.png)

### Design

In designing our adaptive laboratory evolution (ALE) experiment, we consulted Dr. Kim Wise who recently collaborated with a researcher from the Palsson Lab at UC San Diego on an ALE project. From learning how the Palsson Lab automated their adaptive laboratory evolution project, we proposed our evolution project to be initially set at a bottleneck temperature in which cell growth is significantly hindered but present. The SP4 media contains phenol red which changes the color of the media from red to yellow when the media becomes acidic. This can be correlated to cell growth and concentration because minimal cells excrete lactic acid which decreases media pH. We would passage at a ratio of 1 part cell culture to 10 part fresh media when the SP4 media turns yellow. When overnight color change occurs at this passage ratio, we will increase the growth temperature to a new bottleneck temperature that slows the growth rate again.

![build](https://static.igem.wiki/teams/4921/wiki/icons/chemistry.png)

### Build

Starting in December, we began our thermal adaptive laboratory evolution (TALE) project. We started at 42°C, after observing significantly hindered growth rate of minimal cells at this temperature, and continually propagated our cells when the media turned yellow. When overnight growth at 42°C was observed, we determined a new bottleneck temperature using a temperature gradient experiment run with PCR tubes and a thermocycler. We also had multiple lines of minimal cells evolving at the same time to increase the likelihood of beneficial mutations appearing and being selected out.

![test](https://static.igem.wiki/teams/4921/wiki/icons/magnifying-glass.png)

### Test

After a little under 4 months of continual evolution, we obtained strains of minimal cells that can grow overnight at 44.2°C. This is tested through culturing these evolved minimal cells using a 1:10 passage ratio in a waterbath that is measured to be 44.2°C water bath and observing the media change from red to yellow overnight. Aside from obtaining a strain of minimal cell that can optimize the activity of an implemented POAP cycle, we also extracted gDNA of both our evolved minimal cells and ancestral minimal cells. These gDNA were sent off for sequencing in the hopes of identifying the genes and mutations that give rise to thermal tolerance.

![learn](https://static.igem.wiki/teams/4921/wiki/icons/cancer.png)

### Learn

From this project overall, we have learned the techniques of running an adaptive laboratory evolution and how this experiment can lead to the development of desired phenotypes. Over the progress of TALE, we also improved our protocol to overcome challenges. For a majority of time, TALE experiments were ran in eppendorf tubes and the low headspace posed a higher risk of contamination. In response, we began conducting the experiment instead in 5 mL tubes with much greater headspace. Our switch from using heat blocks to waterbaths also allowed our cultures to maintain a more uniform and steady temperature throughout the experiment. We are also in the process of characterizing genes and mutations that are responsible for thermal tolerance in the minimal cell. This characterization work is planned to eventually result in a manuscript that investigates thermal tolerance in the minimal cell and Mycoplasma in general.

References:

[1] Collins, M., Knutti, R., Arblaster, J., Dufresne, J-L., Fichefet, T., Friedlingstein, P., Gao, X., Gutowski, W. J., Johns, T., Krinner, G., Shongwe, M., Tebaldi, C., Weaver, A. J., Wehner, M. F., Allen, M. R., Andrews, T., Beyerle, U., Bitz, C. M., Bony, S., & Booth, B. B. B. (2013). Long-term Climate Change: Projections, Commitments and Irreversibility. In T. F. Stocker, D. Qin, G-K. Plattner, M. M. B. Tignor, S. K. Allen, J. Boschung, A. Nauels, Y. Xia, V. Bex, & P. M. Midgley (Eds.), Climate Change 2013 - The Physical Science Basis: Contribution of Working Group I to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change (pp. 1029-1136). (Intergovernmental Panel on Climate Change). Cambridge University Press.

[2] Santos Correa S, Schultz J, Lauersen KJ, Soares Rosado A. Natural carbon fixation and advances in synthetic engineering for redesigning and creating new fixation pathways. J Adv Res. 2022 Jul 30:S2090-1232(22)00165-5. doi: 10.1016/j.jare.2022.07.011. Epub ahead of print. PMID: 35918056.

[3] Lu Xiao, Guoxia Liu, Fuyu Gong, Huawei Zhu, Yanping Zhang, Zhen Cai, and Yin Li (2022). A Minimized Synthetic Carbon Fixation Cycle. ACS Catalysis, 12(1), 799-808. DOI: 10.1021/acscatal.1c04151

[4] Marian BreuerTyler M EarnestChuck MerrymanKim S WiseLijie SunMichaela R LynottClyde A HutchisonHamilton O SmithJohn D LapekDavid J GonzalezValérie de Crécy-LagardDrago HaasAndrew D HansonPiyush LabhsetwarJohn I GlassZaida Luthey-Schulten (2019) Essential metabolism for a minimal cell eLife 8:e36842.

[5] Pieulle L, Guigliarelli B, Asso M, Dole F, Bernadac A, Hatchikian EC. Isolation and characterization of the pyruvate-ferredoxin oxidoreductase from the sulfate-reducing bacterium Desulfovibrio africanus. Biochim Biophys Acta. 1995 Jul 3;1250(1):49-59. doi: 10.1016/0167-4838(95)00029-t. PMID: 7612653.

[6] González-Cabanelas D, Wright LP, Paetz C, Onkokesung N, Gershenzon J, Rodríguez-Concepción M, Phillips MA. The diversion of 2-C-methyl-D-erythritol-2,4-cyclodiphosphate from the 2-C-methyl-D-erythritol 4-phosphate pathway to hemiterpene glycosides mediates stress responses in Arabidopsis thaliana. Plant J. 2015 Apr;82(1):122-37. doi: 10.1111/tpj.12798. PMID: 25704332.

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### All **Experimental Protocols** can be found within the PDF.

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\*In the interest of conciseness, we will detail the notable and important milestones of our project. There are countless experiments that we ran, especially during our troubleshooting of different primer combinations and E. Coli plasmid assembly, and we feel that attaching every result will only clutter this page. For the full comprehensive journey, which includes every experiment and result we performed for this project, please visit our [Notebook](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%20%7D%7D) tab where we documented every step of this project!

# Design Results

Our project is initiated by the screening and selection of appropriate variants of genes to build the pyruvate carboxylase (PYC), oxaloacetate acetylhydrolase (OAH), acetate-CoA ligase (ACS), and pyruvate synthase (PFOR) (POAP) cycle. In the report of the POAP cycle, researchers used variants of genes that yielded impressive carbon fixation rates at 50 degrees Celsius [1]. This operating condition is too hostile for the minimal cell (JCVI-Syn3B) and is hostile for many potential biological hosts. To overcome this obstacle, we aimed to find different variants of enzymes that constitute the POAP cycle, compare their characterized activities with regards to temperature and pH, and build a POAP cycle that is more suitable for biological conditions (< 45 degrees Celsius). Amongst the 4 enzymes that complete the POAP cycle, the biggest challenge is from PFOR which requires high operating temperatures to overcome the thermodynamic barriers of pyruvate synthesis. Using Braunschweig Enzyme Database (BRENDA), we searched through characterized enzymes from all facets of life and compared their reported activities in association with temperature and pH, yielding the following selection. It is worth noting that while some enzymes had multiple variants selected, our project eventually focused on the testing of one particular variant (bolded), and the other selections became backups in the case of failure.

| Gene | **Organism** | Organism Type | Working Temp (°C) | Gene Length (nt) |
| --- | --- | --- | --- | --- |
| PFOR | **Desulfocurvibacter africanus** | Bacteria | 40 | 3699 |
| PYC | **Bacillus subtilis** | Bacteria | 37 | 3443 |
| OAH | **Aspergillus niger** | Fungus | ≤45 | 1026 |
| OAH | Moniliophthora perniciosa | Fungus | 30 | 993 |
| OAH | Dianthus caryophyllus | Plant | 20 | 1267 |
| ACS | Dunaliella tertiolecta | Algae | 37-45 | 2464 |
| ACS | **Pseudomonas putida** | Bacteria | 38 | 1962 |
| ACS | Bacillus subtilis | Bacteria | 40 | 6300 |

Figure 1.1: Table of selected genes to build a POAP cycle that is more compatible with the growth conditions of the minimal cell (JCVI-Syn3B).

After selection of desired variants of enzymes, we designed fragments of DNA and attached it to the selected genes. The first fragment is a complementary region to the pCC1BAC EcoRV fixed HindIII Exp Vector backbone, a copy control vector that we are using to integrate our genes of interest into the minimal cell. This complementary fragment allows our gene of interest to anneal onto the backbone and be assembled into the plasmid via Gibson assembly and E. Coli plasmid assembly. The second fragment is a mycoplasma promoter of intermediate strength. This allows our genes to be expressed once integrated into the minimal cell. Additionally, we also designed primers to PCR, and crossover PCR, our genes of interest which allows us to generate large quantities of desired genes and confirm its fidelity post plasmid assembly. The crossover PCR procedure is necessary because certain designed gene fragments such as ACS and PFOR were too large so we can only order them to be synthesized in 2 and 3 pieces respectively. After benchling designs, these parts were ordered for synthesis by Twist Biosciences. These components have also been registered as basic and composite parts. Please see the list of these parts in our Parts section of the wiki.

Amongst the 4 enzymes that build the POAP cycle, PYC from Bacillus subtilis was already integrated into the pCC1BAC EcoRV fixed HindIII Exp Vector and transformed into the minimal cell from a previous J. Craig Venter Institute (JCVI) project. This transformed minimal cell containing PYC is supplied to us by the Suzuki Lab at JCVI. Thus, we did not need to synthesize and transform this gene of interest into E. Coli and the minimal cell but had access to its designs nonetheless.

![](https://static.igem.wiki/teams/4921/wiki/results/gene-design-and-plasmid-design/backbone.png)

Figure 1.2: pCC1BAC EcoRV fixed HindIII Exp Vector backbone.

![Syn_PpACS](https://static.igem.wiki/teams/4921/wiki/results/gene-design-and-plasmid-design/synacs-2.png)

![Syn_PFOR](https://static.igem.wiki/teams/4921/wiki/results/gene-design-and-plasmid-design/synpfor-2.png)

![syn_AnOAH](https://static.igem.wiki/teams/4921/wiki/results/gene-design-and-plasmid-design/synoah-2.png)

Figure 1.3: Composite DNA for ACS, PFOR, and OAH genes containing vector complementary regions for plasmid assembly and mycoplasma promoter for expression in minimal cells.

![iGEM_AnOAH](https://static.igem.wiki/teams/4921/wiki/results/gene-design-and-plasmid-design/oahplasmid.png)
![iGEM_PycA](https://static.igem.wiki/teams/4921/wiki/results/gene-design-and-plasmid-design/pycplasmid.png)
![iGEM_PpACS](https://static.igem.wiki/teams/4921/wiki/results/gene-design-and-plasmid-design/acsplasmid.png)
![iGEM_PFOR](https://static.igem.wiki/teams/4921/wiki/results/gene-design-and-plasmid-design/pforplasmid.png)

Figure 1.4: Plasmid design of OAH, PYC, ACS, and PFOR genes assembled into the pCC1BAC EcoRV fixed HindIII Exp Vector.

# Experimental (Build) Results

Upon arrival of our parts, we first performed PCR on composite parts containing genes of interest to both obtain larger quantities of it and to prepare for E. Coli plasmid assembly. It is important to point out that designed gene fragments such as ACS and PFOR were too large so Twist Bioscience divided ACS and PFOR fragments into 2 and 3 pieces respectively. Before moving onto E. Coli plasmid assembly, we first performed crossover PCR to combine the two fragments of ACS into one and the three fragments of PFOR into one.

![Ordered Gene PCR](https://static.igem.wiki/teams/4921/wiki/results/gel/ordered-gene-pcr.png)

Figure 2.1: Agarose gel image showing the PCR products of our composite parts.

![ACS Crossover PCR](https://static.igem.wiki/teams/4921/wiki/results/crossover-pcr/acs-crossover-pcr.png)
![PFOR Crossover PCR](https://static.igem.wiki/teams/4921/wiki/results/crossover-pcr/pfor-crossover-pcr.png)

Figure 2.1.2: Two Agarose gel showing successful assembly of Pseudomonas putida (Pp) ACS fragments and Desulfocurvibacter africanus (Da) PFOR from their fragments using crossover PCR.

---

After obtaining large stocks of our composite parts containing genes of interest, we began the integration of our genes into the plasmid vector backbone through E. Coli plasmid assembly. This assembly works similarly to the Gibson Assembly (fun fact, Gibson Assembly is developed at J. Craig Venter Institute (JCVI) by Daniel Gibson - a JCVI Professor!) but occurs directly inside of E. Coli (please see our Notebook and Experiments for the protocol). Although there was a process of trial and error, we eventually obtained 3 plasmids containing ACS, OAH, and PFOR respectively. E. Coli colonies with successful assemblies of the complete plasmid gains chloramphenicol resistance and appears on a chloramphenicol plate. These colonies were then picked, upscaled, and mini-prepped to confirm plasmid fidelity. It is worth noting that we are assembling individual genes into distinct constructs to ensure that each component works and as a proof of concept. If we assemble all 4 genes into one large construct, it will be immensely more difficult to troubleshoot when encountering an issue.

![ACS_lb_plate](https://static.igem.wiki/teams/4921/wiki/results/lb-plates/acs.png)
![OAH_lb_plate](https://static.igem.wiki/teams/4921/wiki/results/lb-plates/oah-png.png)
![PFOR_lb_plate](https://static.igem.wiki/teams/4921/wiki/results/lb-plates/pfor-png.png)

Figure 2.2: Transformed E. Coli colonies growing on a chloramphenicol plate. These E. Coli colonies successfully assembled our gene of interest into the vector backbone and are hence transformed. On the left is E. Coli with ACS in the plasmid. In the middle is E. Coli with OAH in the plasmid. On the right is E. Coli with PFOR in the plasmid.

---

![ACS plasmid gel](https://static.igem.wiki/teams/4921/wiki/results/gel/acs-plasmid-gel.png)
![PFOR-OAH plasmid gel](https://static.igem.wiki/teams/4921/wiki/results/gel/pfor-oah-plasmid-gel.png)

Figure 2.3: Plasmids obtained from mini-prepping transformed E. Coli culture. On the left gel, the labels 9.1, 9.2, 9.3, 9.4 are 4 separate colonies picked from the plate containing E. Coli cultures with the ACS gene inside of the plasmid. On the right gel are plasmids obtained from E. Coli colonies containing the PFOR gene inside of the plasmid, 2 separate colonies picked from the plate containing Aspergillus niger OAH, 2 separate colonies picked from the plate containing Moniliophthora perniciosa OAH, and the pCC1BAC EcoRV fixed HindIII Exp Vector backbone.

In Figure 2.3, we observe that the plasmids are heavy and are the size we expect them to be. On the gel containing PFOR and OAH variants, we see that the vector backbone has two bands with one heavier than the other. This is likely due to supercoiling of the vector which, based on whether the plasmid is supercoiled or not, separates the two forms of plasmid on an agarose gel. While this is unpleasant to look at, we simply wanted confirmation that our assembled plasmids have sizes within expectation which is indeed confirmed form these results.

---

After having some confirmation that our plasmids are present and correctly assembled, we sent these plasmids for sequencing by Plasmidsaurus. This is also the point at which we prioritized testing for just one variant of a gene for future steps, and plasmids containing other variants of the same gene were stored as a backup.

## AnOAH Sequencing Results

![AnOAH_Sequencing](https://static.igem.wiki/teams/4921/wiki/results/sequencing/anoah-plasmid-sequence.png)

Figure 2.4: Sequenced plasmid containing OAH (SD2c1) aligned with benchling plasmid design (pCC1BAC\_EcoRV\_fixed\_HindIII\_Exp\_Vector\_file2\_AnOAH). The OAH gene is present in the assembled plasmid from nucleotide 2285 to nucleotide 3325 which aligns perfectly with the benchling design, showing that our plasmid is correctly assembled according to our initial design.

## DaPFOR Sequencing Results

![PFOR_Sequencing_1](https://static.igem.wiki/teams/4921/wiki/results/sequencing/pfor-plasmid-seq-1.png)
![PFOR_Sequencing_2](https://static.igem.wiki/teams/4921/wiki/results/sequencing/pfor-plasmid-seq-2.png)
![PFOR_Sequencing_3](https://static.igem.wiki/teams/4921/wiki/results/sequencing/pfor-plasmid-seq-3.png)
![PFOR_Sequencing_4](https://static.igem.wiki/teams/4921/wiki/results/sequencing/pfor-plasmid-seq-4.png)

Figure 2.5: Sequenced plasmid containing PFOR (SD1c1) aligned with benchling plasmid design (pCC1BAC\_EcoRV\_fixed\_HindIII\_Exp\_Vector\_file2\_PFOR). The PFOR gene is present in the assembled plasmid from nucleotide 2285 to nucleotide 5983 which aligns perfectly with the benchling design, showing that our plasmid is correctly assembled according to our initial design.

## PpACS Sequencing Results

![ACS_Sequencing_1](https://static.igem.wiki/teams/4921/wiki/results/sequencing/acs-plasmid-seq-1.png)
![ACS_Sequencing_2](https://static.igem.wiki/teams/4921/wiki/results/sequencing/acs-plasmid-seq-2.png)

Figure 2.6: Sequenced plasmid containing ACS (SD9c1) aligned with benchling plasmid design (pCC1BAC\_EcoRV\_fixed\_HindIII\_Exp\_Vector\_file2\_PpACS). The ACS gene is present in the assembled plasmid from nucleotide 2285 to nucleotide 4246 which aligns perfectly with the benchling design, showing that our plasmid is correctly assembled according to our initial design.

---

After confirming the fidelity of both our gene of interest and our plasmid through sequencing and alignment with our benchling designs, we proceed to transformation of the minimal cell. What is particularly interesting about this step is that once the plasmid enters the minimal cell, it becomes a suicide plasmid that can no longer replicate. However, our plasmid vector contains a cre-loxp site. The same cre-loxp site is also engineered into the genome of the minimal cell which is known as the landing pad. Our gene of interest, along with an antibiotic resistance gene, will be integrated into the genome of the minimal cell before the plasmid is degraded. Thus, successful transformations will yield minimal cell colonies on an antibiotic SP4 plate in which the plasmid is no longer present, but the gene of interest (and an antibiotic resistance gene) it carries is inserted into the genome of the minimal cell and can be expressed.

![posneg_sp4_plate](https://static.igem.wiki/teams/4921/wiki/results/sp4-plates/pos-neg-ctrl-sp4-plate.png)
![oahacs_sp4_plate](https://static.igem.wiki/teams/4921/wiki/results/sp4-plates/acs-oah-sp4-plates.png)
![pfor_sp4_plate](https://static.igem.wiki/teams/4921/wiki/results/sp4-plates/pfor-sp4-plate.png)

Figure 2.7: SP4 antibiotic agar plates that selects for minimal cells that have integrated a gene of interest and antibiotic resistance gene into their genome from the suicide plasmid. On the left, a positive and negative control plate is used to ensure that our protocol works correctly. We expect no colonies to form in the No-DNA side because no antibiotic resistance gene is supplied. We expect colonies to form in the pSG023 side as that is a control vector that has been shown to deliver an antibiotic resistance gene into the minimal cell genome. In the middle, the 9.2 side are minimal cell colonies that now have the ACS gene. The 4.3 side are minimal cell colonies that now have the OAH gene. On the right, the 2.1 and 1.1 side are both minimal cell colonies that now have the PFOR gene. The difference in 2.1 and 1.1 is just that the plasmids were mini-prepped from two separate E. Coli colonies that both have assembled the PFOR plasmid.

---

As mentioned above, because our genes of interest are integrated into the minimal cell genome and the plasmids are soon eliminated/degraded, we lose the ability to mini-prep our minimal cells and send the results off for sequencing. Instead, we use PCR with primers we have designed to both see if a product is produced and if that product is of the expected size. Successful PCR products of the expected size are sufficient for us to be confident in our integration of the genes of interest into the minimal cell and proceed to activity screening.

![minimal cell PCR](https://static.igem.wiki/teams/4921/wiki/results/gel/pcr-post-minimalcelltransformation.png)

Figure 2.8: PCR products obtained from performing PCR on transformed minimal cells. 2.1 are PCR products obtained from minimal cells containing the PFOR gene. 4.3 are PCR products obtained from minimal cells containing the OAH gene. 9.2 are PCR products obtained from minimal cells containing the ACS gene. VC is vector control which affirms that PCR of our transformed colonies yield a product that is heavier, showing that our gene of interest is present.

# GC-MS

After confirming that our minimal cells now have genes of interest contained in their genome, we move on to screening of enzyme activity. Our enzymes do not contain tags which allow for protein purification and SDS-PAGE analysis. Instead, we wanted to rely on gas chromatography-mass spectrometry (GC-MS) to screen for metabolic perturbations. All of these enzymes perturb the central metabolism of the minimal cell, and because the minimal cell has the simplest metabolism that is very well characterized, we plan on using the GC-MS to compare the metabolic profiles of a normal minimal cell and metabolic profiles of transformed minimal cells. Knowing exactly what each enzyme does, we also have a general expectation of what kind of perturbation will occur and to which metabolites it will affect. To better find our target metabolites in the profile, we also created standards of pyruvate, acetate, acetyl-CoA, and oxalate, though not all of them worked as expected (discussed later).

## Pyruvate Standard Profile by GC-MS

![GC-MS oxalate overview](https://static.igem.wiki/teams/4921/wiki/results/gc-ms/oxalate-standard/ovw.jpg)

Figure 3.1: Pyruvate standard (derivatized and dissolved in solution of pyridine, methoxylamine, and MSTFA) profile by GC-MS.

## Derivatized pyruvate elution time and predicted structure

![gcms pyruvate elution time](https://static.igem.wiki/teams/4921/wiki/results/gc-ms/pyruvate-standard/elution-time.jpg)
![gcms pyruvate identity](https://static.igem.wiki/teams/4921/wiki/results/gc-ms/pyruvate-standard/identity.jpg)

Figure 3.2: Derivatized pyruvate elution time (top image), mass-spectrometry (top image), and predicted structure (bottom image).

## Oxalate Standard Profile by GC-MS

![GC-MS pyruvate overview](https://static.igem.wiki/teams/4921/wiki/results/gc-ms/pyruvate-standard/oview.jpg)

Figure 3.3: Oxalate standard (derivatized and dissolved in solution of pyridine, methoxylamine, and MSTFA) profile by GC-MS.

## Derivatized pyruvate elution time and predicted structure

![gcms oxalate elution time](https://static.igem.wiki/teams/4921/wiki/results/gc-ms/oxalate-standard/oxalicaicd.jpg)
![gcms oxalate identity](https://static.igem.wiki/teams/4921/wiki/results/gc-ms/oxalate-standard/oxalicacid-i.jpg)

Figure 3.4: Derivatized oxalate elution time (top image), mass-spectrometry (middle image), and predicted structure (bottom image).

---

The development of standards makes it much easier to screen for specific metabolites in the minimal cell profile as we will know exactly when our target compound elutes and what the derivatized structure will look like. The key metabolites that will be directly perturbed by the enzymes involved in the POAP cycle include acetyl-CoA, pyruvate, oxaloacetate, oxalate, and acetate. We discovered that derivatization using pyridine and methoxylamine does not work well with acetyl-CoA and acetate since the GC profile of these standards show no product (all visible peaks are from pyridine, methoxylamine, and MSTFA). We are working actively to find another derivatizing agent that can work well with these metabolites. Concurrently, we did not have access to oxaloacetate which is a rather uncommon and unstable (typically stored at -20 Celsius) compound. We have placed an order for oxaloacetate and will make a standard for it after arrival.

For the time being, our GC-MS screens of transformed minimal cells are limited to PFOR and PYC genes. Both OAH and ACS lack critical standards for screening - oxaloacetate, acetate, and acetyl-CoA. Still, we collected GC-MS data for all of our transformed minimal cells (including OAH and ACS) but manually searching for derivatized oxaloacetate, acetate, and acetyl-CoA proved difficult.Therefore, we mainly conducted GC-MS screens and analysis on PFOR and PYC genes, and will revisit the collected profiles of OAH and ACS after making appropriate standards (we have attached their profiles in Supplemental Materials). We adapted an untargeted metabolite derivatization and screening protocol from plant natural products to use on the minimal cell, and obtained the following profiles for normal minimal cells and transformed minimal cells.

## Minimal Cell Standard Profile by GC-MS

![GC-MS minimal cell overview](https://static.igem.wiki/teams/4921/wiki/results/gc-ms/base-s3b/ovw.jpg)

Figure 3.5: Metabolic profile of untransformed minimal cell by GC-MS.

## Derivatized pyruvate elution time and predicted structure

![gcms minimal cell elution time](https://static.igem.wiki/teams/4921/wiki/results/gc-ms/base-s3b/pyruvate.jpg)
![gcms minimal cell identity](https://static.igem.wiki/teams/4921/wiki/results/gc-ms/base-s3b/la.jpg)

Figure 3.6: Derivatized pyruvate and lactic acid profile of untransformed minimal cell by GC-MS.

metabolite extraction and derivatization protocol we used provided a comprehensive coverage of biological compounds. Similarly, the GC-MS method we developed, with the help of our PI Dr. Immo Burkhardt, also reinforces this comprehensive coverage of biological compounds. In Figure 3.5, we were able to find metabolites such as pyruvate and lactic acid in the earlier elution phase. Further down the profile, we find a collection of amino acids. Near the middle and end of the profile, we find lipids and large organic molecules. Finally, near the end, we find cholesterol. This coverage of polar and primary metabolites to amino acids then to non-polar lipids and cholesterol gives us confidence that our derivatization and GC-MS is holistically capturing the metabolic landscape of our cell. Further, our profile contains little to no noise and contaminants, allowing us to cleanly distinguish between metabolites and even capture compounds with small concentrations. For a more detailed illustration of the metabolites captured in Figure 3.5, including which peak corresponds to what compound, their mass spectrometry, and the predicted structure using mass spectrometry, please visit our supplementary materials.

## GC-MS standard profile for minimal cell transformed by PFOR

![GC-MS PFOR transformed minimal cell overview](https://static.igem.wiki/teams/4921/wiki/results/gc-ms/pfor/ovw.jpg)

Figure 3.7: Metabolic profile of PFOR transformed minimal cell by GC-MS.

## Derivatized pyruvate elution time and predicted structure for the minimal cell transformed by PFOR

![gcms PFOR transformed minimal cell elution time](https://static.igem.wiki/teams/4921/wiki/results/gc-ms/pfor/pyruvate.jpg)
![gcms PFOR transformed minimal cell identity](https://static.igem.wiki/teams/4921/wiki/results/gc-ms/pfor/la.jpg)

Figure 3.8: Derivatized pyruvate and lactic acid profile of PFOR transformed minimal cell by GC-MS.

## GC-MS standard profile for minimal cell transformed by PYC

![GC-MS PYC transformed minimal cell overview](https://static.igem.wiki/teams/4921/wiki/results/gc-ms/pyc/ovw.jpg)

Figure 3.9: Metabolic profile of PYC transformed minimal cell by GC-MS.

## Derivatized pyruvate elution time and predicted structure for the minimal cell transformed by PYC

![gcms PYC transformed minimal cell elution time](https://static.igem.wiki/teams/4921/wiki/results/gc-ms/pyc/pyruvate-ms-undetected.jpg)
![gcms PYC transformed minimal cell identity](https://static.igem.wiki/teams/4921/wiki/results/gc-ms/pyc/la.jpg)

Figure 3.10: Derivatized pyruvate and lactic acid profile of PYC transformed minimal cell by GC-MS.

---

For PYC and PFOR, we are focusing on observing the perturbation to pyruvate and lactic acid because these are the metabolites that the two enzymes will perturb directly and indirectly, respectively. Lactic acid relates to pyruvate in the minimal cell because the minimal cell converts pyruvate into lactic acid which is a dead-end metabolite. Thus, we expect the levels of lactic acid to change in response to changes in levels of pyruvate. PYC converts pyruvate into oxaloacetate, so we expect a decrease to pyruvate and lactic acid levels. PFOR should facilitate pyruvate synthesis, so we expect an increase to pyruvate and lactic acid levels. The area under the curve, or integration of an elution peak, tells us the abundance of a metabolite profiled by GC-MS. The integration of all of our profiles have been compiled in tables below.

| Sample | Cell Pellet Mass (mg) | Pyruvate Area | Lactic Acid Area | Pyruvate Area per mg of cell pellet mass | Lactic Acid Area per mg of cell pellet mass |
| --- | --- | --- | --- | --- | --- |
| Base Syn3B | 0.8 | 44270.99 | 800901.88 | 55338.7375 | 1001127.35 |
| Syn3B-PFOR | 0.7 | 31699.12 | 1033966.48 | 45284.45714 | 1477094.971 |
| Syn3B-PYC | 1.1 | 13599.86 | 283049.79 | 12363.50909 | 257317.9909 |

Table 3.1: Cell pellet mass, integration of pyruvate and lactic acid profile, and normalization of pyruvate and lactic acid integration for minimal cell samples.

| Sample | Percent change of pyruvate area per cell pellet mass compared to Base Syn3B | Percent change of lactic acid area per cell pellet mass compared to Base Syn3B |
| --- | --- | --- |
| Syn3B-PFOR | -18.2% | +47.5% |
| Syn3B-PYC | -77.7% | -74.3% |

Table 3.2: Percent change of normalized pyruvate and lactic acid profile integrations of transformed minimal cells compared to the base (untransformed) minimal cell.

From Table 3.2 we see that PFOR transformed minimal cell (Syn3B) did not increase pyruvate levels, but it did greatly increase levels of lactic acid by nearly 50%. While this seems conflicting, we believe this still makes sense because we can imagine the minimal cell attempting to stabilize an increase to pyruvate levels by directing more pyruvate to be converted into other metabolites, including the one of the main waste products produced from pyruvate - lactic acid. Thus, the impressive increase to lactic acid levels may allude to the pyruvate synthesis reaction catalyzed by PFOR. Because we are not attempting to characterize the activity rates of PFOR and are merely looking for some sign of perturbation that gives us confidence to assemble the pathway, we feel this is sufficient for us to incorporate this PFOR into the POAP pathway assembly. While the PFOR data may not be definitive, we feel that the PYC transformed minimal cells give us high confidence in proceeding to the next step. We expect decreases to both pyruvate and lactic acid, and this indeed what we observe - a quite significant decrease to both metabolic concentrations.

---

While the data generated from transformed PFOR may not be as convincing as transformed PYC, we are reasoned to proceed with the construction of the big plasmid containing all four enzymes. What would be a true deal breaker would be insignificant perturbation to the metabolic profile compared to the untransformed minimal cell or the inability for transformed minimal cells to grow and proliferate, none of which are observed. Thus, we feel that there is a proof of concept and have proceeded with the assembly of the POAP pathway into one plasmid.

# Construction of POAP Pathway (Current Progress and Future Directions)

The cloning of an enzyme gene downstream of another enzyme gene turned out to be difficult, possibly due to the end sequences not suitable for homologous recombination in E. coli. We devised a cloning method to combine enzyme genes one at a time using crossover PCR and the self-closure of the generated PCR products within E. coli. So far, we have completed the construction of a 2-gene construct (OAH and ACS), confirmed through agarose gel and soon to be sequenced by Plasmidsaurus. We are conducting the final assembly soon to have a 4-gene construct (PYC, PFOR, OAH, and ACS). Subsequent to this, we will introduce the construct into the JCVI-Syn3B cell, confirm the introduction using PCR, and analyze the metabolites produced using GC-MS. Despite our ongoing work, we have demonstrated a proof of concept that the POAP pathway can be implemented in vivo within the minimal cell.

![2 gene construcs OAH and ACS](https://static.igem.wiki/teams/4921/wiki/results/gel/2-gene-construct-oah-acs-gel-2.png)

Figure 4.1: Agarose gel showing the assembly of a 2 gene construct containing OAH and ACS. The first lane from left is the plasmid containing OAH. The second lane from left is the plasmid containing both OAH and ACS. We observe that the band in the second lane is heavier than the one in the first lane, indicating that it is a 2-gene construct. The ladder is the lane on the furthest right and the other lanes are irrelevant.

# Thermal Adaptive Laboratory Evolution (TALE)

An ongoing project parallel to the work on carbon fixation is an adaptive laboratory evolution (ALE) project aimed at evolving thermal tolerance in the minimal cell (JCVI-Syn3B). The goal of this ALE experiment is to complement the POAP cycle because of reports that show the POAP cycle, especially the PFOR enzyme, exhibiting higher activity at higher temperatures ( > 45 Celsius). The ancestral/normal minimal cell grows optimally at 39 degrees Celsius and can not grow at this temperature, but we planned to evolve thermal tolerance through continual adaptive laboratory evolution. After a little under 4 months of continual evolution at bottleneck temperatures, we have obtained strains of the minimal cell that grows overnight at 44.2 degrees Celsius. On one hand, we successfully obtained strains of minimal cells more compatible with the POAP cycle. On the other hand, we were intrigued by the genetic changes that gave the evolved minimal cells the ability to grow overnight at a temperature that would kill the ancestral strain. Thus, we have collected cell pellets of our thermal tolerant minimal cell and sent them for gDNA extraction and sequencing by Plasmidsaurus. We report our sequencing results and preliminary mutation callouts below.

![TALE summary](https://static.igem.wiki/teams/4921/wiki/results/tale/d8p1-ancestral.png)
![TALE summary](https://static.igem.wiki/teams/4921/wiki/results/tale/d8p2-tale.png)

Figure 5.1: Returned gDNA sequencing results of ancestral (unevolved) minimal cells and thermal ALE minimal cells. The left image is the ancestral minimal cell. The right image is the thermal tolerant (evolved) minimal cell.

![TALE summary](https://static.igem.wiki/teams/4921/wiki/results/tale/ale-mutation-callouts.png)

Figure 5.2: Mutations present in the thermal ALE minimal cells but not found in the ancestral minimal cells. This work is ongoing.

Our success with ALE of the minimal cell has yielded us a more compatible host for the POAP cycle which we will soon transform into, once the POAP pathway has been assembled. At the same time, this project opens up new research directions in the discovery of genes and mutations that are responsible for thermal tolerance. Further, the evolution of a synthetic and minimal cell is always exciting as we can learn and compare the evolution of primitive life forms. In summary, the genetic analysis of this ALE experiment is ongoing and will be continually spearheaded by the JCVI-UCSD iGEM team concurrent to carbon fixation. We, and our PIs, envision this to eventually fruition into a publication.

# Mini Review

In our literature search, there was little literature that consolidates information on carbon fixation and synthetic biology to aid researchers in the development of in vivo carbon fixation and cellular carbon capture. To help future researchers tackle the climate crisis using synthetic biology approaches, we drafted a mini-review article that will be submitted for review in Frontiers: Bioengineering and Biotechnology. We have attached a snippet of our manuscript.

![mini review](https://static.igem.wiki/teams/4921/wiki/results/mini-review.png)

Figure 6.1: Snippet of our manuscript which will be submitted for review to Frontiers in Bioengineering and Biotechnology as a mini-review by the time of 2023 iGEM Jamboree.

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