Team:NTNU Trondheim/Data

From 2011.igem.org

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= Data =
= Data =
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== rrnB P1 promoter ==
 
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[[File: Primer_rrnBL_060711_resultat.JPG|thumb|PCR products from rrnB P1 BioBrick separated on 1.5 % agarose. The marked products represent rrnB P1 with regular pre/sufix]]
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==Our System==
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[[File:NTNU2011 system.png|center|]]
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The rrnB P1 promoter is negatively regulated by ppGpp [Kilde] and therefore sutible for our stress sensor.
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==Our favorite new parts==
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We found the
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promoter in the registry distribution (BBa_K112118) submitted by Berkly in 2008.
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This part is in the BBb standard form [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard]
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#[http://partsregistry.org/Part:BBa_K639003 Parts registry] - '''Stress sensor, BBa_K639003.''' The complete fluorescent stress sensor construct.
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and is not compatible with the parts in the BBa standard. In order to get the part in BBa standard we
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#[http://partsregistry.org/Part:BBa_K639000 Parts registry] - '''Stress sensor precursor, BBa_K639000.''' This brick will yield red colonies as it is. When a promoter is inserted in front of the brick, the red color will disappear or be weaker, due to production of LacI that repress lac-promoter.
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PCR amplified the promoter using the BBa_K112118 as template and primers containing the BBa prefix and suffix:
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#[http://partsregistry.org/Part:BBa_K639002 Parts registry] - '''rrnB, BBa_K639002.''' rrnB P1 promoter with standard prefix and suffix in BBa format.
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[[File: Test_rrnB_C3_060811_wiki.jpg|thumb|rrnB P1 promoter in the psb1C3 plasmid]]
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==Characterization of Pre-existing Parts==
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#[http://partsregistry.org/Part:BBa_K112118:Experience Parts registry] - '''rrnB P1, BBa_K112118.''' rrnB P1 promoter BioBrick in BBb format made by the Berkley iGEM team in 2008.
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#[http://partsregistry.org/Part:BBa_K292006:Experience#User_Reviews Parts registry] - '''LacI repressor, BBa_K292006.''' LacI repressor that SupBiotech-Paris iGEM team made in 2009.
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{| border="1"
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==Other parts==
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!Primer
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!Type
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!Sequence
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|rrnB P1 F
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|Forward
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|GTTTCTTCGAATTCGCGGCCGCTTCTAGAGACGTATCCTACGCCCGTGGT
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|-
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|rrnB P1 R
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|Reverse
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|GTTTCTTCCTGCAGCGGCCGCTACTAGTACGCCTTCCCGCTACAGAGTCA
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|}
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The PCR-product was run on a 1,5% agarose gel in order to verify that we had achieved the correct product and to separate it from any other unwanted products.
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The new rrnB P1 biobrick was inserted in the psb1C3 plasmid provided by the iGem HQ.
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Plasmid from five colonies were digested with the enzymes BstBI and SpeI
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It seems like parallel 1 and 2 have the insert.
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== Stress Sensor ==
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[[File: Super_BB__minus_p1_1-5-2.jpg|thumb|Five parallels digested with… ]]
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The stress sensor consist of MCherry (a RFP gene) controlled by the Lac promoter and the Lac inhibitor (LacI) controlled by the rrnB P1 promoter.
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The MCherry biobrick (BBa_J06702) is complete with both RBS and a terminator sequence, and we fist connected this part with the LacP biobrick BBa_R0011.
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The LacI biobrick BBa_C0012 had a RBS region but no terminator, so we added the term biobrick BBa_B0015.
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Then we connected these to constructs to get the final stress sensor minus the P1 promoter.
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In order the check if the construct was correct we digested the plasmid with ........ 
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[[File:construct_map_1807.jpg|thumb|Plasmid map of the complete construct. Promoters are shown in blue, and genes as inside arrows. Only single restriction sites are shown.]]
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Then we added the P1 promoter using the PCR-product as insert. To check if the part was inserted we digested the resulting plasmids with BstBI cutting in the promoter region.
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#[http://partsregistry.org/Part:BBa_K639001 Parts registry] - '''relA, BBa_K639001.''' relA codes for ppGpp synthase, which has been shown to be useful for regulating the rrnB P1 promoter, mimicking the stringent response when overexpressed.
{{:Team:NTNU_Trondheim/NTNU_footer}}
{{:Team:NTNU_Trondheim/NTNU_footer}}

Latest revision as of 07:37, 21 September 2011



Data

Our System

NTNU2011 system.png

Our favorite new parts

  1. [http://partsregistry.org/Part:BBa_K639003 Parts registry] - Stress sensor, BBa_K639003. The complete fluorescent stress sensor construct.
  2. [http://partsregistry.org/Part:BBa_K639000 Parts registry] - Stress sensor precursor, BBa_K639000. This brick will yield red colonies as it is. When a promoter is inserted in front of the brick, the red color will disappear or be weaker, due to production of LacI that repress lac-promoter.
  3. [http://partsregistry.org/Part:BBa_K639002 Parts registry] - rrnB, BBa_K639002. rrnB P1 promoter with standard prefix and suffix in BBa format.

Characterization of Pre-existing Parts

  1. [http://partsregistry.org/Part:BBa_K112118:Experience Parts registry] - rrnB P1, BBa_K112118. rrnB P1 promoter BioBrick in BBb format made by the Berkley iGEM team in 2008.
  2. [http://partsregistry.org/Part:BBa_K292006:Experience#User_Reviews Parts registry] - LacI repressor, BBa_K292006. LacI repressor that SupBiotech-Paris iGEM team made in 2009.

Other parts

  1. [http://partsregistry.org/Part:BBa_K639001 Parts registry] - relA, BBa_K639001. relA codes for ppGpp synthase, which has been shown to be useful for regulating the rrnB P1 promoter, mimicking the stringent response when overexpressed.


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