Team:Caltech/Week 3

Monday 6/28

• Tested the success of the construct we made using digest and gel electrophoresis.
  • Digested ligation product from 6/26 with EcoRI and PstI.
  • Ran digests on gel along with 1kb and 100kb molecular ladders.
• Gel result: No bands were visible other than those of the ladders. Amount of DNA used was most likely too low and thus the ligation product could not be confirmed.

Image of a test gel of our third ligation product. Note that the middle two lanes are sadly empty.

• Transformed additional BioBricks from 2010 Spring DNA Distribution into DH5alpha cells
  • K274100, K274200, K274002, K274003, K274004

Tuesday 6/29

• Transformation results: Transformation of the ampicillin-resistant BioBricks seemed to be successful. However, no bacterial growth was present in the plates and liquid cultures with tetracyclin present. This seems to indicate a problem with the tetracyclin used.
  • Of the plates with bacterial growth (K274100 (red), K274200 (orange)), there was evidence of pigment production, indicating a successful transformation.
• Transformed 7 more bricks (pigments & our AND gate components):
  • K274110, K274210, K274003, K274004, C0076, C0077, R0077, I1765001

Wednesday 6/30

• Transformation results: Only two plates did not have colonies on them.
  • The liquid cultures of the red and orange dyes, K274110 and K274210, seemed to be different colors than the other cultures. When centrifuged down, the cells showed signs of pigment production.
  • Both the colonies and the liquid culture of K274004 (light green) were darkly colored. There was pigment production; however, the pigment did not appear to be a light green color.
• Prepared glycerol stocks.
• Retransformed three bricks and one new brick (GFP):
  • I765001 K274004 C0077 I13504

Thursday 7/1

• Transformation results: All transformations were successful! There was no problem with the antibiotic used.
  • K274003 (dark green) was a dark gray color - evidence of production of pigment.
  • The colonies of I13504 (GFP reporter gene) did not fluoresce under UV light.
• Performed minipreps and created glycerol stocks from the liquid cultures.
• Received requested bricks from the Registry of Standard Parts. We plated and created liquid cultures of the cells containing the bricks.
• Began digests of bricks according to the NEB BioBrick assembly kit protocol.
  • KI765001 (upstream)
  • KI13504 (downstream)
  • pSB1C3 (plasmid backbone)
  • B0034 (upstream)
  • K274100 (downstream)
  • pBAD18 plasmid backbone (including an AraC operon)
• Began ligation reactions:

1. KI765001/KI13504/pSB1C3

• Attaching the UV promoter to a GFP reporter will allow us to test the promoter.

1. B0034/K274100/pBAD18

• To be used for a pigment-production experiment in minimal media.

• Transformed:
  • Ligation 1 & 2 products (above)
  • R0011
  • J23119
  • R0080

Friday 7/2

Note: Friday is an Institute Holiday (which explains why we didn't do much today).

• Mini-preped & made freezer stocks of the following bricks:
  • R0011, J23119, R0080, J04450, K112806, J13002, I737020, K274001, K215000, J63010.
  • Only Ligation 2 was successfully transformed.

Weekend 7/3-4