Adapting the Chassis

Of the four enzymes within the POAP pathway, pyruvate synthase places a thermal constraint on the operating conditions of the minimal cell. Pyruvate synthase operates in the correct direction within the POAP pathway at 45°C. This poses a problem for the minimal cell because a normal minimal cell cannot withstand temperatures above 37°C. Instead of looking for variants of pyruvate synthase that could operate at lower temperatures, the JCVI-UCSD team opted to conduct thermal adaptive laboratory evolution (TALE) of the minimal cells. TALE is conducted by serially passaging minimal cell cultures in slowly increasing temperatures. As a result of this process, we would be picking mutant minimal cell cultures that could grow at higher temperatures. These cultures would be continuously pressured to grow at higher temperatures until the minimal cell could withstand the operating conditions of pyruvate synthase. Through TALE, we have successfully evolved the John Craig Venter Institute’s Syn 3.0B minimal cell to become thermally tolerant at a temperature of 45.2°C. Sequencing results of the thermally tolerant minimal cells pushes for the conclusion that the cells did evolve specific mutations for heat tolerance and not adaptations.

Implementing the POAP Pathway

We are introducing the POAP pathway into the minimal cell created by the J. Craig Venter Institute. This minimal cell would be this thermally tolerant variant to accommodate the high operating temperature of pyruvate synthase, a key enzyme in the POAP pathway. Ultimately, we designed MACS, a minimal and adapted carbon sequestration cell, using the JCVI minimal cell as the biological chassis and the POAP pathway as the inserted synthetic carbon fixation pathway. We will also report the selection of POAP genes based on temperature and pH constraints, assemble all genes into minimal cell plasmids via Escherichia coli assembly, and the purification and sequencing of assembled plasmids. Further, we will soon begin in-vivo activity screens using GC-MS and LC-MS depending on the metabolites observed to assess success of POAP implementation into the minimal cell.

Our current design solution is to implement a 2-Enzyme plasmid design, consisting of two plasmids, labeled PFOR-ACS, which consists of the ACS gene inserted into the PFOR gene-containing backbone, and PYC-OAH, which consists of the OAH gene inserted into the PYC gene-containing backbone. This design is particularly advantageous in that it allows us to test and analyze one half of the POAP plasmid at a time.

An additional boon of this design is that it is easier to engineer than our initial 4-Enzyme design since we can deal with a much smaller backbone when inserting our genes. With the 4-Enzyme plasmid, it was difficult to get past the 3-Enzyme stage due to the sheer size of the 3-Enzyme vector. 

One potential drawback of this design that must be considered is that we are skipping over the steps that produce acetyl-CoA and oxalate. By not knowing how these two products are affected we may not be able to confirm unexpected effects on the metabolic growth of the minimal cell. Oxalate, in particular, is toxic. If we analyze the PYC-OAH transformed strain of the minimal cell and find that it is not able to produce the perturbations in metabolic compounds that we are expecting, the presence of oxalate presents a confounding factor, as it could be affecting cell growth. 

At current stages, most of the work relies on performing cloning procedures in order to ensure successful completion of each 2-Enzyme plasmid. To build the 2-Enzyme design, the gene for the insert (ACS and OAH) must be inserted into the already constructed single-enzyme backbone (PFOR and PYC, respectively). Homologous regions on each insert that correspond to their respective backbone were built up to 50 base pairs for the upstream homology region and 200 base pairs for the downstream homology region using sequential cycles of PCR. In order to build up the regions of homology, several PCR runs had to be performed with slightly different forward primers for each insert. Each PCR run would allow a short sequence of new nucleotides matching the backbone to be added to the upstream end of the genetic fragment. ACS required 2 consecutive PCR runs and OAH required 7. These runs were confirmed successful using gel electrophoresis. The backbones were linearized using enzyme digestion, HindIII for PYC and EcoRI for PFOR. The pieces were then bound together using Gibson Assembly and transformed into DH5-alpha E. coli cells for amplification, all of which proved successful using Plasmidsaurus sequencing.

After successfully confirming the correct building of the plasmids using Plasmidsaurus sequencing, next steps involve the transformation of these plasmids into the minimal cell. 

After transformation, further procedures and analysis will be performed on the novel 2-Enzyme strains in order to determine whether the product does accurately fit the specifications and constraints of the minimal cell design. This analysis will involve either liquid or gas chromatography-mass spectrometry in order to quantify successful enzyme catalyzation. 

Making a Minimal Media 

The original media used to grow our minimal cells was SP4 media. SP4 media is an expensive, complex media. It is used to culture the minimal cell because it is highly nutritious and created for mycoplasma, which is what the minimal cell is evolved from. Recognizing the cost that this could have on research efforts, we began optimizing a defined media to grow the minimal cells in.

Our defined media called C5 media. C5 media contains 51 ingredients and as a defined media, those ingredients and their concentrations are all known. In the development of C5 media, current efforts are focused on reducing the number of components by eliminating those not necessary for cell growth. C5 media contains a supplement called CMRL, which testing has determined to be necessary for cell growth, as seen in Figure 10. The inclusion of phenol red in the media allows for visual confirmation of cell growth, since our minimal cell secretes lactic acid during growth. The orange color of the cell culture with CMRL in the image indicates cell growth while the pink color of the cell culture without CMRL in the image indicates no cell growth. The test was performed by formulating a stock of C5 media without CMRL. Minimal cell cultures that had been previously evolved to grow in C5 media were thawed and cultured in C5 media with and without CMRL. The CMRL was added to the aliquoted media used for cell culture in the same ratio that it would typically be added to the stock. The two cell culture lines were then incubated at 37 degrees Celsius. The cell culture with CMRL grew overnight, confirming the ability of the cells to grow in C5 media. The cell culture without CMRL did not grow after several days of incubation, confirming that CMRL is necessary for cell growth. The chemical composition of CMRL is known, so future research will be focused on determining which components of CMRL are necessary for cell growth through single-elimination experiments.

The development of a cost effective media will prove monetarily beneficial to this project. Although the costly additive CMRL was proven to be necessary for cell growth, the composition of CMRL is known, so future research can be done to determine which exact components are necessary. Because the CMRL compound is the bulk of the cost in this cell culture media, the reduction of the unnecessary components in the future research can reduce overall costs of minimal cell growth and maintenance.

Page Leader: Ella Kirwan