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
  • [http://partsregistry.org/wiki/index.php?title=Part:BBa_K274100 K274100], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K274200 K274200], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K274002 K274002], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K274003 K274003], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K274004 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 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K274100 K274100 (red)], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K274200 K274200 (orange)]), there was evidence of pigment production, indicating a successful transformation.
• Transformed 7 more bricks (pigments & our AND gate components):
  • [http://partsregistry.org/wiki/index.php?title=Part:BBa_K274110 K274110], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K274210 K274210], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K274003 K274003], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K274004 K274004], [http://partsregistry.org/wiki/index.php?title=Part:BBa_C0076 C0076], [http://partsregistry.org/wiki/index.php?title=Part:BBa_C0077 C0077], [http://partsregistry.org/wiki/index.php?title=Part:BBa_R0077 R0077], [http://partsregistry.org/wiki/index.php?title=Part:BBa_I1765001 I1765001]

Wednesday 6/30

• Transformation results: Only two plates did not have colonies on them.
  • The liquid cultures of the red and orange dyes, [http://partsregistry.org/wiki/index.php?title=Part:BBa_K274110 K274110] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K274210 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 [http://partsregistry.org/wiki/index.php?title=Part:BBa_K274004 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):
  • [http://partsregistry.org/wiki/index.php?title=Part:BBa_I765001 I765001] [http://partsregistry.org/wiki/index.php?title=Part:BBa_K274004 K274004] [http://partsregistry.org/wiki/index.php?title=Part:BBa_C0077 C0077] [http://partsregistry.org/wiki/index.php?title=Part:BBa_I13504 I13504]

Thursday 7/1

• Transformation results: All transformations were successful! There was no problem with the antibiotic used.
  • [http://partsregistry.org/wiki/index.php?title=Part:BBa_K274003 K274003 (dark green)] was a dark gray color - evidence of production of pigment.
  • The colonies of [http://partsregistry.org/wiki/index.php?title=Part:BBa_I13504 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.
  • [http://partsregistry.org/wiki/index.php?title=Part:BBa_KI765001 KI765001] (upstream)
  • [http://partsregistry.org/wiki/index.php?title=Part:BBa_KI13504 KI13504] (downstream)
  • [http://partsregistry.org/Part:pSB1C3 pSB1C3] (plasmid backbone)
  • [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0034 B0034] (upstream)
  • [http://partsregistry.org/wiki/index.php?title=Part:BBa_K274100 K274100] (downstream)
  • pBAD18 plasmid backbone (including an AraC operon)
• Began ligation reactions:

1. [http://partsregistry.org/wiki/index.php?title=Part:BBa_KI765001 KI765001]/[http://partsregistry.org/wiki/index.php?title=Part:BBa_KI13504 KI13504]/[http://partsregistry.org/Part:pSB1C3 pSB1C3]

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

1. [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0034 B0034]/[http://partsregistry.org/wiki/index.php?title=Part:BBa_K274100 K274100]/pBAD18

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

• Transformed:
  • Ligation 1 & 2 products (above)
  • [http://partsregistry.org/wiki/index.php?title=Part:BBa_R0011 R0011]
  • [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23119 J23119]
  • [http://partsregistry.org/wiki/index.php?title=Part:BBa_R0080 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:
  • [http://partsregistry.org/wiki/index.php?title=Part:BBa_R0011 R0011], [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23119 J23119], [http://partsregistry.org/wiki/index.php?title=Part:BBa_R0080 R0080], [http://partsregistry.org/wiki/index.php?title=Part:BBa_J04450 J04450], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K112806 K112806], [http://partsregistry.org/wiki/index.php?title=Part:BBa_J13002 J13002], [http://partsregistry.org/wiki/index.php?title=Part:BBa_I737020 I737020], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K274001 K274001], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K215000 K215000], [http://partsregistry.org/wiki/index.php?title=Part:BBa_J63010 J63010].
  • Only Ligation 2 was successfully transformed.

Weekend 7/3-4