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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:
- [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.
- [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
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