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 4C, 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 100uL 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 100uL 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.

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 DH5\alpha 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]

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.