Notebook

Day-to-Day Lab Details

5/26/23

• Prepared the plasmid DNA retrieved from PCR testing to be sent off to a separate lab for Sanger Sequencing and also introduced that DNA into S17 strain E. coli cells through transformation.

5/30/23

• Four plates from transformation failed; redid those 4 plates for transformation. The rest of the plates were successful so we continued onto mating S17 strain E. coli and methanotrophs, using NMS mating media that creates an environment where both can grow with the goal of the target DNA transferring from the E. coli to the methanotrophs. However, since the S17s had little biomass, we restreaked them on new plates for continued growth and for a 2nd mating process. We also made new LB and NMS mating media for bacteria to grow on.

6/6/23

• Met with team colleagues to work on applying for grants and reviewing our research in order to prepare for a presentation with our team manager.

6/7/23

• Worked further on presentation topics and on applying for grants with team colleagues.

6/8/23

• Same as previous day.

6/9/23

• Worked further on presentation, collaborated with team colleagues to prepare to analyze results from methanotrophs, and had meeting with professor on progress made for the week

6/12/23

• Met with team to give updates on progress and discussed plans for the week as well as the remainder of the summer. Also analyzed methanotroph cultures after 1 week of growth.

6/13/23

• Prepared DH10B strain E. coli cells in liquid culture to be analyzed for fluorescence. Results were inputted in an Excel sheets and graphs were created accordingly.

6/14/23

• Preperation of LB Gent/Kan Plates as well as NMS selection and mating media.
• Restreaking of s17 competent cells to prepare for mating.

6/15/23

• Mating of s17's with bath.

6/16/23

• Restreaked E.coli and methanotroph mating plates on NMS Selection media. Worked with team to finalize responses for grant.

6/20/23

• Flourimeter measurement of Anderson Promoter activity in Bath.
• Colony PCR and data entry for the flourimeter results.

6/21/23

• Preperation of agragose gel to confirm colony PCR results, DNA cleanup for sequencing, but results were unsatisfactory so we decided not to send off for sequencing.
• Dishes, plastics, other lab upkeep.

6/22/23

• Made LB Gent and mating media. Restreaked S17 E. coli on LB Gent media for mating the following day with OB3b strain of methanotrophs.

6/23/23

• Mated the S17 E. coli that grew overnight with OB3b methanotrophs and placed the cultures in 30 C incubator to grow over the weekend.

7/12/23

• Not applicable labwork.

7/13/23

• Not applicable labwork

7/17/23

• Second fluorescence measurements in liquid culture bath. Started overnight liquid e. coli cultures.

7/18/23

• Fluorescence measurement replicate in e. coli, data entry. Restreaked s17's to prepare for mating with Ob3B. Made Gen10 NMS selection plates for after matings, made mating plates for mating with Ob3B.

7/19/23

• What we did here

7/20/23 - 9/16/23

• This is the time period where the mutagenesis work was done, mostly by student instructor Spencer Lee.