Part:BBa_K515106:Design

J23103 promoer - RBS B0034-RFP E1010 - Holin K112805 - endolysin K112806

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 616
  Illegal AgeI site found at 728
  Illegal AgeI site found at 1730
  Illegal AgeI site found at 1800
  
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

This part has been made to comply to the following specifications for Gene Guard:

1. Prevent horizontal gene transfer by making any other cell that is not our own GMO non-viable

• Horizontal gene transfer (Figure 1) was the main problem that we could think of when it came to releasing GMOs in field tests and later in the implementation stage of the project. This is a common issue even in GM crops where cross-pollination between the GMO and natural organisms is a constant worry. In bacteria, genes are usually transferred to other species through conjugation or by the uptake of the genetic material from lysed GMOs. Since the genetic material can still be taken up by other species after the lysis of our GMO, a traditional kill switch where one uses an input to induce lysis is not a viable and safe option. Therefore, we thought that the device must be present within the genetic material itself and induce a response in the naturally occurring bacterium instead.

2. Must be able to test whether the system works

• To do this we must use reporter genes. The most common reporter genes are fluorescent proteins like RFP and GFP. Since the CRIM plasmid already has sfGFP, we will be using that to test for the expression of anti-holin. In order to test whether the plasmid is functional, we will be integrating RFP into the plasmid. We hope to be able to see a natural bacterium that has taken up the plasmid fluoresce red before lysing by widefield microscopy.

Source

genomic sequence