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 - Got a working prototype of the program working and chose a standard vector for protein purification (pET28+). Showed that the plasmid could be linearized with the primers that were ordered (they seemed iffy).

This week - Scale up the program and make primers for all components that we have now once protein purification is shown to work with the vector we are using (waiting on Eric for results). Start cloning the constructs out of E. coli and put them in vectors for protein purification. Figure out how to handle modeling/find out what to present for the Caltech meeting. Though at the risk of sounding like someone who is all talk no action, I'd like to have a joint meeting with all interested in the modeling aspect of the project to figure out what is tractable for the Caltech meeting and REU as well as what would be most useful to researchers working with the PURE system currently.

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: rebuilding the 50S subunit of the ribosome. This includes synthesizing its different parts: r-proteins, coenzymes, 5S rRNA and 23S rRNA, the latest being the most challenging.
• Put together latest work on ribosome biogenesis - putting together the latest information available on ribosome biogenesis
• Synthesizing primers for ribosomal genes - Alec is working to create a program that allows us to create primers quickly and effectively since we will be creating a large quantity of primers
• Other - Learning wetlab skills: PCR, Primer design, Protein Purification, Gibson assembly, Bacterial transformation, Competent cell preparation