BAC Weekly Task List
List of planned work for the week for each individual on team
- Last week - Progress
- This week
Week of 8/7/17
Last week - Tested the prototype of the program and generated multiple (10) primers for PURE parts. Tested the PCR once and all failed; will retry to see if failure is due to primer design or experimental setup. Also made a list of reactions that occur in PURE in preparation to build the PURE model.
This week - Re-PCR the 5 components that I have designed via the program and see if amplification occurs. Build the model of PURE based on the Katy Wei thesis to be presentable at the Build-A-Cell meetup in Pasadena.
- Reconstitution of E. coli RNA polymerase in PURE - All templates for 5 subunits have been cloned into plasmid and linear templates, reconstituted E. coli RNAP with plasmid templates works with nanoluciferase assay
- Characterization of E. coli RNA polymerase in PURE - Ran successful PURE reactions with nanoluciferase and GFP linear templates, but mRFP-Spinach reporter is still non-functional. Will debug PCR for mRFP-Spinach linear templates with 25-bp handles.
- Liposome formation - Working on reconstitution of "outer solution"/solution A of PURE.
- Protein purification - Will learn the procedure from Eric this week.
- Purify IF1 and IF2 - still cloning, but have ValS + others to test
- Clone 4 vectors for PURE proteins - Have 8 in progress
- Continue purification of PURE components
- Reconstitute Sol A and verify using commercial PURE kit
- Last week: Collection of materials for testing Bacillus subtilis minipreping and storage. Tested conjugation with Vibrio natriegens.
- This week: Preparing DNA synthesis sequences.
- Successfully transformed the conjugative plasmid pRK24 into recipient strain MG1AC53.
- Next step on this confirming the presence of pRK24 with gel.
- Read literature on no-SCAR system with the purpose of replacing the oriT-kan casette with negative selection marker SacB on pRK24. Design appropriate sequences to carry out this experiment. This would be done to avoid back-conjugation of the genome from the recipient to the donor.
- SUMMER PROJECT: reconstructing the 50S subunit of the ribosome. This includes synthesizing its different parts, expressing them in PURE, and coupling it with native 30S to text for functionality.
- Put together latest work on ribosome biogenesis - constantly looking for updates
- Synthesizing primers for ribosomal genes - working with Alec to create a program that allows us to create primers specific to create our vector (would need it for over 110 genes)
- "This week": continue to purify more ribosomal components into vector and get them to express in pure + look for other labs that se an automated method of cloning as opposed to monocloning