Michigan State iGEM’s research begins with questions regarding the three layers of our Comprehensive Biocontainment System, Synterception:
- How has GhOST been characterized and how is it an effective Toxin-Antitoxin system
- How can we characterize and target foreign conjugative plasmids?
- How efficiently can Synterception remediate baseline conjugation?
|Toxin Antitoxin System||Baseline Conjugation Assay and Modeling|
To characterize the GhoST system, we wanted to create a two vector system to determine the relative levels of expression of GhoT that would kill the cells, as well as the amount of GhoS that would “rescue” the cells and allow them to grow in the presence of GhoT. We decided to use two vectors developed by Dr. Jonathan Silberg’s lab, pSac16 and pFd031 , which are two compatible vectors with different inducible promoters. We could then separately induce GhoT and GhoS in the same cell transformed with both vectors. We first designed two gBlocks for each vector that when assembled together, would form the complete plasmid. One of the gBlocks for pSac16 was the GhoT toxin fused to mCherry, and on one of the gBlocks for pFd031 was the GhoS antitoxin fused to GFP. We decided to attach fluorescent markers to the toxin and antitoxin to be able to qualitatively measure the levels of expression of both proteins. This way, we could determine how much GhoT expression was needed to kill the cells, as well as how much GhoS expression relative to GhoT was needed to allow the cells to grow. For each vector, we designed one of the gBlocks to have homologous overhangs to the other gBlock, so Gibson Assembly could be used to form the complete plasmid. However, this design method was unsuccessful in generating the assembled plasmids, so we later ordered the pFd031 and pSac16 plasmids from Addgene and designed new gblocks to insert into them, in a more traditional cloning style.
Our initial design with the two gBlocks did not work to assemble the vectors. Once we decided to order the full plasmids from Addgene, we amplified the backbone of the pFd031 plasmid and then performed Gibson Assembly with the new gBlock containing the GhoS gene fused to GFP. We then transformed our plasmid into heat competent E. coli DH5⍺. In the end, we were only able to build the antitoxin vector and transform it into our cells.
Unfortunately, we were unable to test our constructs, as the assembly of the vectors was never completed. We planned to characterize the GhoST system by having E. coli containing the GhoT vector, the GhoS vector, and cells that had both vectors present. We planned to add varying levels of the inducers of both the toxin and antitoxin to the different cells in a plate reader. We would measure the OD600 to determine the cells’ death due to the toxin, as well as the growth allowed when “rescued” by the antitoxin. We would also measure the fluorescence produced, as this was indicative of both GhoT and GhoS expression, to determine the relative levels of the toxin that would induce death, as well as the levels of the antitoxin that would inhibit the toxin’s affects.
|In order to test how far to dilute these cultures, plates were divided into 12 sections ranging anywhere from being undiluted, up to 150,000-fold, in order to find what dilutions were necessary to get a countable amount of colonies.|
|Learn||We learned during our time in the lab that our design of two gBlocks to assemble a vector was not working. We were unsuccessful in all of our Gibson Assembly attempts, so we first tried to use another method to assemble the gBlocks together called SLiCE ligation. Unfortunately, we were not able to get this to assemble our vectors either. So, we realized that we had to change our design. We ordered the plasmids Pfd031 and pSac16 from Addgene and designed new gBlocks containing the toxin and antitoxin both fused to their respective fluorescence proteins. This new approach allowed us to obtain the fully assembled pFd031 antitoxin plasmid||Ratio of D:R We found out that the ratio of donor to recipient was not a critical factor when testing conjugation. Dilutions for transconjugants Although testing out a large scale of dilutions for each of our plates was very time consuming, it was crucial for obtaining measurable results and provided us with insight with regards to the amount of transconjugants that will from with a given concentration of donor and recipient cells.|