Team:UGA-Georgia/Notebook

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WET LAB

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Notebook

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.

  • 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] Extracted pMEV4 from bacteria.
  • [E.Coli Lab] PRC for mCherry with 11 mutated RBS sites and 1 native RBS.
  • [E. Coli Lab] Vector pMEV4 was digested with Spe1, Pst1 and buffer 2. Gel Extraction, Ligation and Heat Shock Transformation were also performed.
  • [E. Coli Lab] Colonies were successfully formed from 1,3,4,8, and 11; PCR was redone for 2,5,6,7,10, and 12.
  • [E. Coli Lab] Created new LB plates with amplicillin and LB Broth.
  • [E. Coli Lab] Prepared competent cells for transformation
  • [E. Coli Lab] Obtained PCR product samples 2,5,6,7,10 and 12, and redid vector digestion, gel extraction, ligation, and heat shock transformation.
  • [E. Coli Lab] Sample 5,6,7 and 10 were unsuccessfully transformed. Redid vector digestion, gel extraction, ligation, and heat shock transformation.

  • 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] Screening Part I, Part II for 1,3,4,5,8,9,10,11 and 12. Verification was done by using KpnI and NcoI ( if positive:2750 and 2414; if negative: 2414 and 1986; control: pMEV4).
  • [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 was done for 1,3,4,5,8,9,10,11 and 12. Also made 2 permanent stocks for each of them.
  • [E. Coli Lab] Plasmid extraction, inoculation, and screening were performed on 2, 6 and 7. Also made 2 permanent stocks for each of them


  • 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.