Endy:BAC Subgroup

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BAC Weekly Task List

List of planned work for the week for each individual on team

Suggested format:

  • 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.


Last week:

  • Successfully transformed the conjugative plasmid pRK24 into recipient strain MG1AC53.
  • Next step on this confirming the presence of pRK24 with gel.

This week:

  • 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