We have continued our work on the project “Cloning, Expression, and Purification of the Cellular TORC Protein”. At the end of the fall semester we were working on troubleshooting the process of cloning the TORC2 gene into bacterial cells. We were working on testing the ligase by performing a single digest on both the insert and the vector with one of our enzymes. We saw colonies on the plates with and without ligase, but the plate without ligase has significantly more colonies. This indicates that the ligase does not have an effect and might even be causing a negative effect.
At the beginning of this semester we had to test our DH5-alpha cells to see if they were still competent because the freezer went down over the winter break and competent cells need to remain frozen when they are not being used. Several test transformation was performed with the cells and were all unsuccessful. New competent cells were made and tested with negative results. A second round of cells was successfully made and the cloning process began again. We performed a digestion on the pGEX vector, but we still had digested TORC2 inserts left from the previous semester. We used the digested vector and inserts in a ligation then followed up with a transformation to confirm that the ligase is not working. The results showed no colonies on any of the plates. We will continue to troubleshoot, but will probably need to buy new enzymes or ligase.
Dr. Isaacson had a successfully transformed TORC3 in the pGEX vector, so we moved onto the purification step for TORC3. We then took pGEX-TORC3 and transformed it into Rosetta DE3 pLysS and Rosetta DE3 pLacI competent cells, which should enhance expression of eukaryotic proteins in bacterial cells. We have started test inductions using IPTG to induce protein expression on the TORC3 wildtype and mutants. We have used a Coomassie stain and Western blot to test the TORC3 protein expression and are waiting on those results.
Moving on from here we will continue to try to clone the TORC2 DNA into the bacterial cells, but we will move on with the TORC3 expression step in the meantime.
Once again I am Mikayla Bonnett and Dr. Marisa Isaccson and I are working on the project called Cloning, Expression, and Purification of the Cellular TORC2 Protein. We have been held up working on the cloning step of the process. We have been trying to transform a TORC2 gene from a mammalian vector into an E. coli bacterial vector. This process consists of performing a digestion, a ligation, and then a transformation. Since none of the transformations thus far have been successful, we have been working on troubleshooting our process. At this point we have narrowed it down to the possibility that our ligase or the ligase buffers are not working properly.
We performed a digestion without an insert and only one enzyme so the pGEX-4T2 vector would theoretically ligate back on itself. We then performed two ligations with two different stocks of ligase and this digestion. They were left to sit for an hour and then put in the freezer. The following day the transformations were performed. There were 112 colonies on the plate without ligase, 57 colonies on the 2014 ligase plate, and 72 colonies on the 2013 ligase plate. Since the results showed that more colonies on the plate with no ligase than both the plates with ligase, which shows that either the ligation did not work or the ligase is bad.
We have now decided to test the ligation two more times and if we do not yield any results then we will buy new ligase. We are going to perform the same ligation but perform a transformation after an hour instead of freezing it and performing it the next day. The ligation will be left out of the freezer and also allowed to proceed overnight. If the hour ligation transformation isn’t successful then we will try the transformation one more time to see if letting the ligation proceed overnight has any effect on the results.
Hello, my name is Mikayla Bonnett. I am doing research with Dr. Marisa Isaacson. The title of our project is “Cloning, Expression, and Purification of the Cellular TORC2 Protein”. I am extending my research from summer 2015. Human T Cell Leukemia Virus (HTLV) is a retrovirus that causes leukemia and lymphoma. Tax is a transcriptional activator that is required to activate HTLV gene expression. Studies have shown that the cellular TORC proteins help enhance HTLV gene expression, presumably via direct binding to the Tax protein. Since TORC and Tax are both involved in increased HTLV-1 gene expression, we would like to find the relationship between the two. Our goal is to clone the TORC2 gene so we can produce TORC2 protein, which will allow us to look closely at TORC and Tax interactions.
We are continuing our efforts to transfer TORC2 gene from a mammalian vector into an E. coli bacterial vector. We are still using the wild-type and mutant serine to alanine and serine to aspartate forms of TORC2. Over the summer we were delayed at the transformation stage and are now working to troubleshoot this process. We began with a digestion of each form of TORC2 and the pGEX-4T2 vector with increased amounts of each vector. We are in the process of continuing the isolation and purification of the TORC2 DNA to place into the bacterial vector. After the ligation, DNA gel electrophoresis was performed and the next step is to extract the DNA from the gel. Following the extraction, a transformation will be performed. If the transformation is successful then we will screen the colonies by restriction digestion.
We also decided to test the E. coli competent cells we were using. We transformed pUC19 plasmid vector into the older (8/12) and new (8/27) cells to see if they were performing properly. No colonies were found on the older cells, so we will no longer use these cells. Colonies were found in the newer cells so we know they are working properly and can transform our TORC2 DNA samples when we are finished isolating the DNA. A different low concentration of pGEX-4T2 vector was transformed into E. coli. If colonies result then we will be able to make more vector to increase our supply.
We hope to see increased results with this round of TORC2 and pGEX-4T2 vectors since the concentration is increased. Then we can begin to work with the TORC2 and Tax connection to HTLV-1 gene expression.
Our progress on the Cloning, Expression, and Purification of the Cellular TORC2 Protein project has been delayed at the transformation stage of cloning. At this stage the transformants have to be plated onto antibiotic selection plates. After failing to see colonies on two separate transformations, we troubleshot our process. We discovered that the plates we were using had gone bad. So we repeated the transformation again with new plates. Colonies did not form from this transformation, but we did see colonies for our positive control. We then determined the ligation’s vector and insert concentration. After that we decided to redo the ligation and double the pGEX4T2 vector since we found the vector concentration was low. Another transformation was performed with the new ligation and no colonies formed. Since no colonies have formed we are now making a new batch of digested pGEX4T2 vector to hopefully generate digested pGEX4T2 at a higher concentration. If we do see colonies after this we will move onto the next step and screen the colonies for proper insertion of the TORC2 genes. Since there has been some setbacks we have no new results to show.
I have learned how to perform the beginning steps of cloning which are digestion, gel purification, ligation, and transformation. I have developed my skills and knowledge of laboratory equipment I have used in the past and laboratory equipment that is new to me. Another thing I have learned from this experience is that patience is needed in the laboratory because everything does not automatically work in the lab. Sometimes you have to slightly change the process and repeat steps over and over again.
This project has shown me what goes on in the laboratory and has further sparked my interest into the biology research field. I have enjoyed the work and hope to continue to gain more experience.
The title of our project is Cloning, Expression, and Purification of the Cellular TORC2 Protein. The purpose of our project is to look closely at TORC2 and TAX binding. TAX is a transcriptional activator that is required for HTLV-1 gene expression. TORC2 is a cellular gene that is known to increase HTLV-1 gene expression. We are transferring the TORC2 gene from a mammalian expression vector to a bacterial vector. Our goal is to purify the protein in the bacteria cells, so a pure TORC2 protein can be obtained to test TORC and TAX binding. We are using the wild-type and mutant serine to alanine and serine to aspartate form of TORC2. The phosphorylation state of TORC2 regulates the activity level. Therefore, the objective is to find out which form of TORC2, either the phosphorylated or dephosphorylated state, binds with TAX. Then we could find if TAX binds with the active or inactive TORC2 to determine how TORC2 increases HTLV-1 gene expression.
The method we have used so far to answer our research question is molecular cloning to insert the cellular gene TORC2 into a bacteria expression vector. We used a digestion with restriction enzymes and then a ligation to anneal the TORC2 gene into the new vector. The final step was to do a transformation of the TORC2 DNA into E. coli. The first transformation did not produce any colonies, so we will now try a transformation with positive control plasmids.