iGEM 2016 — Team UGA-Georgia

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# Team:UGA-Georgia/Notebook

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# May

## Week 1

* Aliquots of the primers designed for the spacer and RNA secondary structures were made and labelled for storage.
* Inserts for were created for cloning through PCR modification. The mCherry expression vector containing the wild type ribosome binding site was modified with the respective primers from m01-m15.
* Interlab: Brainstorm ideas for Interlab study
  -2 options→ lyse cells using previous methods using SDS or Triton and measure the fluorescence or fix our cells with formaldehyde and measure fluorescence using flow cytometry and plate reader

## Week 2

* PMEV4 expression vector from the previous season was grown in E. coli to provide enough plasmid for the rest of the season.
* LB broth and ampicillin agar plates were made.
* Interlab: Decided to fix cells with formaldehyde since shipping would be easier and won’t require dry ice.
* Prepared rough draft proposal, contacted biosafety, and list of domestic and international iGEM teams to email

## Week 3

* E. coli and Methanoccus maripaludis wild type were grown in large amounts to create frozen stock for the rest of the season (for homogenous and precise results).
* 15 Inserts and PMEV4 vector were excised by restriction enzymes and ligated.
* Anaerobic plates were prepared
* Interlab: Prepared protocol for both flow cytometry and plate reader

## Week 4

* 15 recombinant plasmids were transformed into E. coli with ampicillin.
* Plasmids were extracted via zyppy plasmid miniprep kit.
* Extracted plasmids were transformed into M. maripaludis wild type stock and plated on anaerobic plates.
* Interlab: Prepare media for inoculation of mutants and pick which mutants we will use for our Interlab study

# June

## Week 1

* Formate media was prepared with additional batches of puromycin and sodium sulfide.
* Isolated colonies for each of the mutants on the plates were inoculated into formate media.
* For the part BBa\_K1939000, insert were PCR amplified, and subsequently digested alongside psB1C3. Overnight ligation was performed. The ligation product were transformed in Top 10 E. coli cells and plated on LB agar with chloramphenicol.
* Interlab: prepared email to iGEM teams and finalized proposal and protocol
* Made a google form for teams to access for inputting their data and submitting any changes they made to the protocol.

## Week 2

* For the colonies that came up in media at an OD600 or higher were aliquoted for freezer storage.
* Positive growth colonies were centrifuged to make cell pellets.
* For the part BBa\_K1939000 that will be submitted, colonies were picked from the chloramphenicol plates and were inoculated in LB broth with chloramphenicol. After overnight incubation, plasmid extraction and verification digests were performed.
* Interlab: Emailed all iGEM teams about study
* Grew up mutants: L2C16, L1C12, L1C18 and Wild Type

## Week 3

* Cell pellets were lysed using lysis buffer and the mCherry extracts were harvested.
* mCherry extracts were exposed to oxygen for maturation.
* For the part BBa\_K1939001, insert were PCR amplified, and subsequently digested alongside psB1C3. Overnight ligation was performed. The ligation product were transformed in Top 10 E. coli cells and plated on LB agar with chloramphenicol.
* Interlab: Conducted experiment; harvested cells, re- suspended in media salt and aliquoted into 1ml samples to ship to teams
* Placed samples in shaker overnight for oxygen exposure and added formaldehyde the next day
* Scheduled date to use flow cytometry and plate reader to measure fluorescence

## Week 4

* For the part BBa\_K1939001 that will be submitted, colonies were picked from the chloramphenicol plates and were inoculated in LB broth with chloramphenicol. After overnight incubation, plasmid extraction and verification digests were performed.
* Began continuation work on the R.B.S project from the previous year by determining which mutants still needed to be characterized.
* Interlab: Measured fluorescence using plate reader and finalized protocol for plate reader

# July

## Week 1

+ For the part BBa\_K1939002, insert were PCR amplified, and subsequently digested alongside psB1C3. Overnight ligation was performed. The ligation product were transformed in Top 10 E. coli cells and plated on LB agar with chloramphenicol.
+ Frozen stock from the previous year’s Ribosome Binding Site project were inoculated in fresh media
+ Fresh Media, Antibiotic and Sodium Sulfide was made
+ Interlab: Measured fluorescence using flow cytometry and finalized protocol for flow cytometry

## Week 2

+ For the part BBa\_K1939002 that will be submitted, colonies were picked from the chloramphenicol plates and were inoculated in LB broth with chloramphenicol. After overnight incubation, plasmid extraction and verification digests were performed.
+ Transformation buffer and Agar Plates were made
+ Transformation of a number of spacer mutants was conducted
+ mCherry cell extracts were taken for the Ribosome Binding Site Library mutants
+ Interlab: Analyzed results from both flow cytometry and plate reader
+ Calculated the fluorescence dividing it by the OD and times the dilution factor

## Week 3

+ For spacer mutations colonies that had successful transformations mCherry aliquots were taken.
+ Gel Electrophoresis was conducted to ensure the mCherry aliquots had mCherry gene.
+ For the part BBa\_K1939003, insert were PCR amplified, and subsequently digested alongside psB1C3. Overnight ligation was performed. The ligation product were transformed in Top 10 E. coli cells and plated on LB agar with chloramphenicol.
+ Interlab: Obtained all addresses for iGEM teams participating in Interlab study
+ Shipped cells to domestic teams

## Week 4

+ For the part BBa\_K1939003 that will be submitted, colonies were picked from the chloramphenicol plates and were inoculated in LB broth with chloramphenicol. After overnight incubation, plasmid extraction and verification digests were performed.
+ mCherry aliquots were left in shaker to allow for fluorophore to mature
+ Fluorescent measurement were taken for samples that were positive
+ Interlab: Was able to contact 2 iGEM China teams and ship samples internationally

# August

## Week 1

- Ribosome Binding Site library mutants were sequenced to determine the exact mutation that they contained
- Interlab: Communicated with teams upon receiving the samples and any problems or questions they had when conducting the experiment
- Modeling: The model was adjusted to allow for formate and acetate uptake.

## Week 2

- All data for both Ribosome Binding Site Library and spacer project was compiled and graphics was compiled.
- Interlab: Started to analyze data of teams that had submitted their results.
- Modeling: The effect of formate as a secondary carbon source on optimal geraniol production and biomass formation was evaluated

## Week 3

- For the part BBa\_K1939004, insert were PCR amplified, and subsequently digested alongside psB1C3. Overnight ligation was performed. The ligation product were transformed in Top 10 E. coli cells and plated on LB agar with chloramphenicol.
- Interlab: Continued to analyze data of teams that had submited their results
- Compared our teams data to others teams data and made figures
- Modeling: The effect of acetate as a secondary carbon source on optimal geraniol production and biomass formation was evaluated

## Week 4

- For the part BBa\_K1939004 that will be submitted, colonies were picked from the chloramphenicol plates and were inoculated in LB broth with chloramphenicol. After overnight incubation, plasmid extraction and verification digests were performed.
- Interlab: Finalized figures and made any additional changes
- Modeling: Data was compiled and examined.

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