Endy:BAC Subgroup

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 7/17/17

Alec

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.

Anton

Conary

Cynthia

• Reconstitution of E. coli RNA polymerase in PURE - Waiting on sequencing primers for two more subunits to come in, will do transformations this week.
• Characterization of E. coli RNA polymerase promoter library - Plasmid for positive control was cloned by Eric over the weekend and all promoters were PCR amplified, will do experiments with purchased E. coli RNA polymerase this week, starting with J23119 promoter.
• Liposome formation - Planning liposome experiments with Texas Red and egg PC

Eric

• Make corrections to initiator tRNA structure and order ultramers - Reading old tRNA papers to figure out potential failure mode for nonfunctional initiator tRNA
• Purification of fmt enzyme - Underwent purification, will run on gel to check if successful

• Clone and purify out 6 more PURE proteins
• Read into potential maturation enzymes necessary for the maturation of the initiator tRNA

Keoni

• Last week: Collection of materials for testing Bacillus subtilis minipreping and storage. Tested conjugation with Vibrio natriegens.

• This week: Preparing DNA synthesis sequences.

Maria

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.

Vicky

• SUMMER PROJECT: reconstructing the 50S subunit of the ribosome. This includes synthesizing its different parts: r-proteins, coenzymes, 5S rRNA and 23S rRNA, the last one being the most challenging.
• Put together latest work on ribosome biogenesis - constantly looking for updates on extra components that we can add for 50S synthesis
• Synthesizing primers for ribosomal genes - working with Alec to create a program that allows us to create primers quickly and effectively since we will be creating a large quantity of primers.

• "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