Team:Caltech/Week 3

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==Monday 6/28==
==Monday 6/28==
Line 13: Line 13:
==Tuesday 6/29==
==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==
==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==
==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==
==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==
==Weekend 7/3-4==
 
 
}}
}}

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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:
  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

 
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