Part:BBa_K112245

{pAH57 promoter}{rbs.xis.int!} inserted into K112995 plasmid

All ccdb parts have been withdrawn from the Registry. Samples of parts containing the ccdB gene cannot be requested. - iGEM HQ

This part was inserted into the K112995 plasmid between ccdB and the attR2 site.

Performing Gateway in vivo was tested using this part and an RFP part flanked by att sites in an entry vector. Three trials showed very high efficiency(transformations using miniprepped material gave thousands of red colonies), and low co-transformation rate(less than 1%). For more information about this part, visit our [http://2008.igem.org/Team:UC_Berkeley/GatewayPlasmid Wiki!]

General Procedure

Below is a flowchart of the general plasmid based Gateway procedure. Due to various combinations of assembly and entry vectors explored, the "Gateway Device" was created to represent the {promoter.xis.int!} sequence with or without the adjoining {rbs.ihfα!}{rbs.ihfβ!} sequence. The "Lysis Device" sequence (which was only tested in the pK112245 assembly vector) was similarly simplified.

The major combinations of assembly + entry vectors are: pK112245 + pBca1256, pK112246 + pBca1256, pK112128 + pK112247, and pK112128 + pK112248. Additionally, pBca1256 with five different sized parts replacing the original mRFP part was also tested with each assembly vector to show the Gateway reaction can be done with parts of all sizes, although this is not shown in the diagram below.

Results

• Control done at 30°C.

• As can be seen above, there is a relatively high rate of cotransformation as seen by the growth of colonies on the Spec plates. The trials done at 30°C had a higher rate of cotransformation likely because there were fewer plasmids resulting from the Gateway reaction.
• In an attempt to reduce cotransformation rates in those grown normally at 37°C, we repeated these experiments, picking more colonies and diluting the purified protein mixture 20x before transformation into MG1061 cells. The results are shown below.

• Although these plates have not been allowed to grow as long and thus are not as bright in color, it can clearly be seen that cotransformation has been successfully eliminated.

The Lysis Device

• The lysis device was inserted into the pK112245, and the experiment repeated. However, only 2 colonies grew, although both were free of cotransformation.

• The entry vectors with the various part lengths replacing mRFP in pBca1256 all sequenced correctly; the attB1 and attB2 sites were confirmed for all experiments. This suggests that the plasmid based gateway reaction with pK112245 and pK112246 assembly vectors works for parts as short as 250bp and as long as 3078bp.

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This part is in BBb Format. It is flanked by BamHI and BglII sites instead of XbaI and SpeI. More information about the BBb Format is available at:

[http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 3242
  Illegal PstI site found at 881
  Illegal PstI site found at 5225
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 4084
  Illegal PstI site found at 881
  Illegal PstI site found at 5225
  Illegal NotI site found at 141
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 212
  Illegal XhoI site found at 5265
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 3242
  Illegal PstI site found at 881
  Illegal PstI site found at 5225
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 3242
  Illegal PstI site found at 881
  Illegal PstI site found at 5225
  Illegal AgeI site found at 3674
  Illegal AgeI site found at 3998
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 762
  Illegal BsaI.rc site found at 4366