Part:BBa_K1189001

TALE-B with a his 6 tag under a lacI promoter

This part was built to allow for the extraction of TALB proteins with the his-tags. This part was also built with J04500, an IPTG inducible promoter with RBS. TALB proteins can specifically bind to DNA.

Fig 1: Shows the repeat variable domain(RVD) of TALEs. These RVDs are very specific and the code of 2 amino acids that bind to each nucleotide has been solved making TALEs very easy to engineer and very specific to their targets

Fig 2: Western blot showing the expression levels of TALE-B with and without Kozak sequences. We show that in the presence of kozak sequence TALE-B is not expressed however once we remove the kozak sequence TALE-B is expressed.

Figure 6: TALE capture assay was done with TALE B ( BBa_K1189001 )and TALE A B-lac fusion ( BBa_K1189031 ) and DNA ( BBa_K1189006 ) with both target sequences. If capture is successful, the B-lac is present in the well giving a colour change from pink to yellow when subjected to benzylpenicillin substrate solution within 20 minutes. The only wells that change colour are the first four wells which contain TALE B, specific DNA, and TALE A B-lac fusion and our positive control wells which are the controls for our fusion TALE A B-lac protein and our positive recombinant b-lac. All our other controls including our test using a non-specific sequence of DNA remained pink . This preliminary characterization data demonstrates that the TALEs are able to bind to DNA with specificity. Additionally it also shows that our system of capturing DNA with two detector TALEs and then subsequent reporting of the DNA’s presence works.

This assay shows that we can capture our target DNA with two detector TALEs with specificity . Additionally, we can report whether that DNA has been captured and is present in the sample, which is a very important concept for our sensor system.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 2654
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]