Team:Caltech/Week 4

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 [http://partsregistry.org/Part:BBa_V1012 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 [http://partsregistry.org/Part:BBa_V1012 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 [http://partsregistry.org/Part:BBa_V1012 V1012] in case we need to retry the competence procedure.
• Tested pigment production of cells containing [http://partsregistry.org/Part:BBa_K274100 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 [http://partsregistry.org/Part:BBa_V1012 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 [http://partsregistry.org/Part:BBa_M30109 M30109] for assembly tomorrow, along with cultures of [http://partsregistry.org/Part:BBa_J04450 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 [http://partsregistry.org/Part:BBa_K274100 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.
  • [http://partsregistry.org/Part:BBa_R0077 R0077] (both upstream and downstream)
  • [http://partsregistry.org/Part:BBa_C0077 C0077] (upstream)
  • [http://partsregistry.org/Part:BBa_C0076 C0076] (upstream)
  • [http://partsregistry.org/Part:BBa_M30109 M30109] (downstream)
• Began ligation reactions
  • [http://partsregistry.org/Part:R0082 R0082]/[http://partsregistry.org/Part:BBa_R0077 R0077]/[http://partsregistry.org/Part:pSB1T3 pSB1T3]
  • [http://partsregistry.org/Part:C0077 C0077]/[http://partsregistry.org/Part:B0015 B0015] (previously digested)/[http://partsregistry.org/Part:pSB1T3 pSB1T3]
  • [http://partsregistry.org/Part:C0076 C0076]/[http://partsregistry.org/Part:B0015 B0015] (previously digested)/[http://partsregistry.org/Part:pSB1T3 pSB1T3]
• Transformed ligation products into DH5alpha cells.
• Created liquid cultures
  • ligation 1 product - [http://partsregistry.org/Part:C0076 C0076]/[http://partsregistry.org/Part:B0015 B0015] (previously digested)/[http://partsregistry.org/Part:pSB1T3 pSB1T3]
  • ligation 2 product - [http://partsregistry.org/Part:C0077 C0077]/[http://partsregistry.org/Part:B0015 B0015] (previously digested)/[http://partsregistry.org/Part:pSB1T3 pSB1T3]
  • ligation 3 product - [http://partsregistry.org/Part:R0082 R0082]/[http://partsregistry.org/Part:BBa_R0077 R0077]/[http://partsregistry.org/Part:pSB1T3 pSB1T3]
  • [http://partsregistry.org/Part:B0015 B0015]
  • [http://partsregistry.org/Part:K124017 K124017]
• 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 [http://partsregistry.org/Part:B0015 B0015] and [http://partsregistry.org/Part:K124017 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.