February
Week 1
Introduction to anaerobic facilities for new members
Week 3
Making of NaS solution.
Making of formate media.
Week 4
Creation of transformation buffer (TB).
First geraniol extraction efficiency test with balch tubes.
March
Week 1
Creation of geraniol standards for gas chromatography (GC) analysis/calibration through serial dilutions.
Week 3
Creation of more formate media.
Creation of general salts solution.
Creation of glycylglycine buffer.
- All of these solution protocols can be found on our protocol page.
April
Week 1
Learning about ribosome binding site library (RBS) and our metabolic Optflux model.
Week 2
Made agar plates
Creation of fresh wild type (WT) stocks.
Week 3
Created diluted puromycin
May
Week 4
Inoculation and revival of frozen stocks.
June
Week 1
Determined the optical density (OD) of the previously revived cultures.
[E. Coli Lab] Extracted pMEV4 from bacteria.
[E.Coli Lab] PRC for mCherry with 11 mutated RBS sites and 1 native RBS.
Week 2
Restriction digestion, purification, and ligation of pMEV4 and PCR products (C1 and C2).
Transformation by heat shock, plated the cells, made an agarose gel and ran the products on it.
Picked colonies from the previous day of plating.
Plasmid extraction, digestion, gel electrophoresis, and verification.
[E. Coli Lab] pMEV4 Digestion, Gel Extraction, Ligation and Heat Shock Transformation were performed.
[E. Coli Lab] Extracted pMEV4 from bacteria.
[E. Coli Lab] Colonies were successfully formed from 1,3,4,8, and 11; PCR was done for 2,5,6,7,10, and 12.
Week 3
Revival of pAW50-mCherry frozen stock.
Revival of S0001 (WT) frozen stock.
[E. Coli Lab] Inoculation, plasmid extraction, digestion and screening were performed on 1, 3,4,5,8,9,10,11 and 12.
[E. Coli Lab] Cloning for 2,6, and 7 in progress
Week 4
Prepared the revived cultures for fluorescence microscopy.
Fluorescence microscopy training.
Anaerobic transformation of RBS libraries 1-12 into M. maripaludis.
Puromycin enrichment of transformants.
Made glycerol stocks of original transformants.
Fluorescent microscopy of transformants/pAW50-mCherry/S0001.
[E. Coli Lab] Plasmid extraction, inoculation, and screening were performed on 2, 6 and 7.
July
Week 1
Took ODs of the native RBS and library 1 and 2 (12; L-1; L-2 respectively).
Plated the 12, L-1, and L-2 cultures.
Dispensed media to test tubes.
Picked colonies form the plates.
Week 2
ODs from last weeks picked colonies were taken.
Made frozen stocks of RBS colonies,
Sub-cultured parent strains.
Researched primary literature on mCherry maturation.
Began developing a protocol for oxygen maturation of mCherry.
Week 3
Made frozen stocks of RBS colonies and sub-cultured parent strains.
Re-subcultured and revive pAW50-mcherry.
Subcultured the parent strains of WT and native RBS (12) into triplicates. The triplicates were wrapped in foil to prevent any photobleaching.
The pAW50-mcherry came up and then all samples were taken to a plate reader to perform fluorescence tests.
[E. Coli Lab] the primers for the negative and positive control group were received; ran PCR on the negative and control groups in order to create a large amount of the DNA.
[E. Coli Lab] PCR amplification for mCherry gene with the negative and positive control.
[E. Coli Lab] Solid media was made.
[E. Coli Lab] PCR was performed on 13, 14 and 15; vector digestion was also performed with Spe1, Pst1 and buffer2.
[E. Coli Lab] Gel electrophoresis was done to confirm digestion of 13-15.
Week 5
Filled out and submitted safety form.
The geraniol extraction efficiency experiment was performed.
- Left the organic phase to dry over night and be resuspended to test for voltility.
Track selection.
August
Week 2
Geraniol synthase containing cells (GS) were inoculated into triplicates of varying concentrations of puromycin.
- The previous geraniol extraction methods were repeated with these new GS samples.
Week 3
Geraniol standards were made to be test on GC/MS.
We did an extraction efficiency test between batch tube vs. separatory funnel.
Testing of geraniol on the GC/MS was done.
Week 4
Cells containing our pMEV4 plasmid with native RBS were inoculated into 5mL of rezasurin free (RF) media with varying levels of puromycin concentration.
ODs were taken of the samples and they were placed into darkness.
Week 5
The cells were prepared for the plate reader as per the first plate reader preparation protocol.
The plate reader results were in conclusion and a new protocol was required.
September
Week 2
A frozen stock of pAW50-mCherry was revived in 5mL of RF media.
Room temperature stocks of 3 colonies of 12 were inoculated into 5mL of RF media.
- All of these will act as parent strains that will then be inoculated and grown in varying conditions to test for the best condition for generating mCherry.
Week 3
The parent strains were inoculated and grown in the varying conditions.
- 37C vs. 30C
- 100mL vs. 5mL media
Some of each of the parent strains were also PCR tested to confirm the presence of the mCherry gene.
- The PCR showed that the mCherry gene was indeed present.
Week 5
ODs of the pAW50-mCherry and the 3 colonies of 12 that were grown in varying conditions were taken and they were all above OD=0.6.
The samples were prepared for overnight oxygen exposure as per the protocol.
The samples were taken to the plate reader to measure fluorescence.
- Fluorescence was measured for all the 12 samples in each condition.
Since this pre-study gave us consistent, positive values for fluorescence, we were able to now refine the oxygen exposure protocol and apply it to characterization of our parts.
- This detailed protocol can be found in our protocol section.
Revival of frozen stocks of cells containing plasmids with our theoretical "perfect" RBS (14), cells containing our theoretical "negative" RBS (15), and each of the 3 colonies of 12. Also, revival of WT with no plasmid were revived from a room temperature stock (negative control).
[E. Coli Lab] PCR Verification of 12C1, 12C2 and 12C3 was done with F12 as forward primer and R as reverse primer.
October
Week 1
The revived parent strains of 14, 15, WT, and 12 C-1,2,3 were inoculated into varying volumes, and in triplicates, to test for a linear relationship of mCherry production to volume of culture which would be determined after the samples were taken to the plate reader.
- The triplicates were grown in 5mL, 25mL, and one single sample each of 12 C-1, 14, and 15 were grown into 100mL as a reference.
[E. Coli Lab] Cloning for BioBrick (Genes: BBa_K1383000{Native RBS; F12 with F12 and R primers}, BBa_K1383001{Theoretical Best RBS; F14 with F14 and R primers}, and BBa_K1383002{Theoretical Worst RBS; F15 with F15 and R primers}; Vector: pSB1C3); PCR result was observed by using gel electrophoresis and UV light.
[E. Coli Lab] Plasmid extraction, purification, and screening were performed on F12A, F12B, F12C, F14A, F14B, F14C, F15A, F15B, and F15C.
Week 2
The oxygen exposure protocol was performed on the triplicates and the results of the experiment can be found on our RBS Library page.
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