Part:BBa_K4649011

SK Primer

This is a primer designed specifically to promote the expression of multiple genes at a cloning site. In our system, we used pBluescript vector pBBR1MCS-2, a mobilizable shuttle and expression vector that can replicate in many Gram-negative bacteria such as E. coli and R. tropici. This part serves as an alternative to pcr in our plasmid, which allows for the detection and identification of the genes cloned into the plasmid through gel electrophoresis. Therefore, this part allows us to ensure that our genes have been properly cloned into the plasmid. We codon optimized this part in Benchling. We obtained this DNA from IDT and did not encounter any problems with obtaining and using this part. This part can be used in any biosafety level laboratory. To figure out how best to transform our plasmid into rhizobia, we practiced transforming the plasmid into rhizobia before we had cloned our genes into the plasmid. For our first attempt, we followed a blue-white screening procedure from the 2014 Hannover team for rhizobium electroporation. To test that our plasmid was functional, we performed a blue-white screening. The intact plasmid contains a functioning LacZ gene, so when we add Xgal and IPTG to the plasmid, it should turn blue. However, when the genes have been inserted into the plasmid and the LacZ gene has been disrupted, the plasmid should turn white. A secondary test that we performed was kanamycin selection. The plasmid without the genes inserted is resistant to kanamycin, and so will not die in the presence of kanamycin. However, the newly inserted genes will disrupt the kanamycin resistance, and the bacteria will die in the presence of kanamycin. This procedure proved successful, showing that our plasmid was functional, and therefore that this primer was functional.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 12
  Illegal SpeI site found at 6
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal SpeI site found at 6
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 12
  Illegal SpeI site found at 6
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 12
  Illegal SpeI site found at 6
  
• 1000
  COMPATIBLE WITH RFC[1000]