Team:Caltech/Week 3

iGEM 2010

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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]

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]

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]

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]

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

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]

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.

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