Submitted by Brittnee Crane for TRIM Protein Research
May 21st– May 27th
The purified plasmid obtained after bacteria transformation with proteins extracted from yeast were nicknamed “yeast preps”. The yeast preps ready to be put through a digestion reaction were digested and gel electrophoresis was performed with each. The gel was analyzed to determine which samples would be sent off for sequencing. Three new yeast colonies were obtained (94,95, and 96), purified for DNA extraction, and transformed into bacteria.
The recombination reactions TRIM6, TRIM31 and TRIM69 (mentioned in the previous week) into pGBK were completed again and bacteria transformations followed. Most of the transformations were successful, so colonies were used to seed cultures. Glycerol stocks were made and additional cultures were prepared for plasmid purification and digestion reactions.
Submitted by Brittnee Crane for TRIM Protein Research
May 14th– May 20th
More yeast colonies were streaked out onto “pizza” plates. `A culture for T6(2)-91 was started for Yeast DNA purification and then the purified plasmid was used in bacteria transformations. Due to a mistake during purification, the transformation was unsuccessful. Cultures were started for both glycerol stocks and yeast purification with T6(2)-5(1), T6(2)-5(2), T6(2)-44, T6(2)-54, T6(2)-69, T6(2)-70, T6(2)-75, and T6(2)-78. The culture for #5(1) was unsuccessful. After yeast DNA purification with all the samples, the plasmids were used to transform bacteria and plate them onto ampicillin agar plates. Ampicillin plates were used as all prey plasmids (unknown proteins) were inserted into pGAD, which contains ampillicin. #91 was cultured again.
The PCR reactions mentioned in the previous week: TRIMs 6, 6*, 31, 31*, 69, and 69* (with *=1 ng of plasmid as template and without=0.5 ng of plasmid as template), went through gel electrophoresis and were ALL successful!! The desired amplicons were extracted from the gel and purified. Gel electrophoresis was performed with the gel purified plasmid and the concentration was estimated for each. The gel purified plasmids were used along with purified pGBK digested with EcoRI and BamHI, to complete recombination into pGBK. Bacteria transformations took place with the recombination reactions twice and were unsuccessful both times, except for one transformation. PGBK-TRIM6 grew a single colony that was obtained and cultured.
Submitted by Brittnee Crane for TRIM Proteins
Kurtiss observed as I finished wrapping up a Yeast two-hybrid experiment. I had already ran through the entire protocol, picked yeast colonies that grew on both double drop out and quadruple drop out agar plates with x-α-Gal (after mate and plate with a hcDNA yeast library), and streaked them out for isolation of a single yeast colonies. 15-cm agar plates were divided like a pizza and the sections were numbered. There were 9 colonies there were selected from plates, cultured for glycerol stocks and cultured for DNA purification. PCR was completed using designed primers and synthetic genes TRIM6, TRIM31 and TRIM69 for template.
Our laboratory studies E3 ubiquitin ligase enzymes. This video puts these E3 enzymes into the context of the ubiquitin enzyme cascade that consists of E1, E2, and E3 enzymes as well as ubiquitin and target proteins. The goal for our lab is to identify proteins that the E3 enzymes interact with to inform future studies and form hypotheses about the potential functional roles of the proteins we are attempting to characterize.
For TRIM Proteins Research
- Review the first to do list and discuss your progress with your group
- Write up a progress report summarizing accomplishments and works in progress (see Hannah’s first report, but streamline it a bit)
- Finish your progress report with a list of goals for this week
- Strategy for achieving goals of the first to do list
- Split team effort towards pushing forward on all possible directions of the flow chart
- Plan to use more of the day that just the morning when team leaders are present
When you clone a gene into pGBKT7 and have confirmed that it is the exact sequence you think it is, the next thing you’ll want to do is transform it into yeast. We have a kit for that and the manual is here: PT1172-1.
Some immediate needs are as follows:
- Make a glycerol stock. Just like bacterial freezing media, yeast freezing media consists of 50% glycerol in the typical
- Test whether the gene causes autoactivation of the Y2H system (see if it grows on yeast media lacking tryptophan (confirms that the plasmid is in the yeast) as well as histidine and adenine (tests whether the gene product alone can lead to reporter gene expression). Growth under these conditions means that you can’t use Y2H for your particular TRIM protein.
Next set of experiments:
- Perform a Y2H screen by mating the yeast with your gene in pGBKT7 with the yeast containing a library of human gene cDNA.
- Perform pairwise Y2H assays by mating the yeast with your gene in pGBKT7 with the yeast containing other TRIM and MAGE genes in pGADT7.
Week 1 Lab Summary (5/7/18-5/14/18)
This week my team and I setup a digest for pGADT7-TRIM5α, from a miniprep done by J. Graff previously in the semester. The digest from this sample was lost due to the addition of a ladder being inserted into the sample. The miniprep sample used for this sample ran out, so we started a new culture of pGADT7 and miniprepped that culture to obtain more plasmid to run a digest. Concentration of the miniprep was low so we are going to start and miniprep another culture of pGADT7-TRIM5a. Another solution is to use the low concentration as template for a transformation into E. coli and to do a miniprep on yielded colonies to obtain a better concentration.
PCR amplification of TRIM8 and TRIM13 was unsuccessful with Human cDNA as the template DNA. It is believed that the incorrect concentration of Template DNAwas used so we nanodropped the sample and redid calculations for the correct amount of cDNA to be used in the Thermocycler PCR protocol.
pTWIST-TRIM5α arrived so we transformed the plasmid into E. coli and then picked an isolated colony and grew a culture in LB broth +amp. The culture was miniprepped and nanodropped. That sample was then used in a digest with EcoRI and NotI and a picture was taken for confirmation that TRIM5α was properly extracted from the pTWIST plasmid.
Previously, we have used a laborious method to identify splice variants of the various TRIM proteins. Moreover, we have not had a good method to quickly figure out what protein isoforms are possible from the splice variants.
Our lives just got easier with the discovery of SpliceMiner. Just go to this website and type in the name of the TRIM you’re working with. To try it out, I tried “TRIM6”. Immediately I was given the information to 10 splice variants and 4 protein isoforms.