Team:Caltech/Week 4

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Monday 7/5

Note: Today was another Institute Holiday.

Tuesday 7/6

  • Hydrolyzed linseed oil. The reaction solution will be characterized on Wednesday.

Wednesday 7/7

  • Attempted to make V1012 (strain CP919, KanR, envZ deficient) electrocompetent:
    • Centrifuged overnight 3mL LB-Kan culture at 4°C, 16000rcf for 10min.
    • Discarded supernatant. Resuspended pellet in 1.5mL ice-cold water. Centrifuged again.
    • Repeated with 0.75mL ice-cold water.
    • Resuspended pellet in 1.5mL ice-cold 10% glycerol.
    • Flash-froze 100μL aliquots in dry ice/ethanol.
  • Transformed light/lysis ligation product from last week into V1012 via electroporation. (Voltage: 2.5V; time constant: 4.5)
    • Plated 100μL on LB-Tet agar plate & inoculated a 5mL LB-Tet overnight culture.
  • Inoculated 5mL LB-Kan culture of purely V1012 in case we need to retry the competence procedure.
  • Tested pigment production of cells containing K274100 in various solutions.
    • Tested solutions of LB, LB without yeast extract added, 80% glycerol, 100% glycerol, and glycerol with different concentrations of agar.
    • Since the brick is under an arabinose-inducible promoter, two versions of each solution were made - one containing arabinose, and one without arabinose.
    • Pigment production was subjectively measured every hour for five hours. It seemed like there wasn't going to be much pigment produced so the solutions were left overnight in the shaker.
  • Obtain the absorbance of hydrolyzed linseed oil using ethanol as blank. The peak shifts toward larger wavelength comparing to pure linseed oil.
  • Test the absorbance of .25wt% agar in glycerol, .5wt% agar in glycerol and water at 632nm. .25 wt% agar gives the lowest absorbance value.

Thursday 7/8

  • The V1012 transformation appears to have worked - the plate is covered in 1000+ colonies.
    • Made freezer stock from parallel liquid culture.
  • Miniprepped the DNA and prepared a sample for sequencing.
  • Started LB liquid culture of M30109 for assembly tomorrow, along with cultures of J04450 to perform a quick lysis test (in preparation for testing the lysis construct).
  • Prepared 2YT and SOB media in preparation for making electrocompetent DH5α cells tomorrow. Also prepared all glassware and other supplies.
  • Completed schematics for our printing process, including a detailed gene network diagram. We will begin assembly of the 3D genetic constructs tomorrow.
  • Results of the pigment test with K274100: None of the cells seemed to have created the red pigment we were looking for.
    • It seems likely that that all the cells died.
    • Did not have a visible pellet of cells in the glycerol and agar solutions after centrifugation. The solutions were probably too viscous.

Friday 7/9

  • Digested bricks according to NEB BioBrick assembly kit protocol.
  • Began ligation reactions
  • Transformed ligation products into DH5alpha cells.
  • Created liquid cultures
  • Background reaction using AIBN
    • Test both lysed and unlysed cell
    • Solvent: .1 ml .5 wt % agar in glycerol
    • Amount of AIBN: 3 wt % with respect to the cell pellet.
    • Reaction is run under theater light for 3 hrs.
    • The unlysed cell appears to be lysed at the end of 3 hrs.

Weekend 7/10-11

  • Made minipreps of B0015 and K124017
  • Transformation results: Transformations failed.
    • There could be a problem with the antibiotic used or there could also be an issue with the digestions and/or ligations.
  • Background reaction using AIBN with more controls
    • After 3 hrs of reaction, transfer a small sample from each reaction into separate 5 ml of LB solutions and leave the solution in the shaker overnight.
    • The sample from the reaction with 3wt% AIBN, glycerol, and cells containing K274004 biobrick produces a slightly cloudy solution with large amount of dark green pigments precipitated at the bottom, which is similar to the sample taken from a lysed solution.
 
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