Difference between revisions of "Part:BBa K1189001:Design"
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===Design Notes===
 
===Design Notes===
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Transcriptor Activator-Like Effectors (TALEs) are proteins produced by bacteria of the genus Xanthomonas and secreted into plant cells. These naturally occurring TALEs play a key role in bacterial infection, as they are responsible for upregulation of the host genes required for pathogenic growth and expansion (Mussolino & Cathomen, 2012). These special bacterial plant pathogen proteins bind to DNA by specifically recognizing one base pair with a single tandem repeat in their DNA-binding domain. TALEs are an advantageous tool in synthetic biology because they can be modified to bind to a chosen DNA sequence.  
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On the iGEM parts registry we found TALB (BBa_K782006) that the Slovenian team synthesized for a previous project. TALEs are very large proteins and take a long time to design and synthesize. Therefore, we decided to use these TALEs to test our system. We also saw this as an opportunity to use and build upon parts made by former iGEM teams. When we sequenced TALB (BBa_K782006), we discovered that it had a small mutated segment. We expected a sequence of AGCAATGGG in the repeat variable di-residue of the second repeat. However, the sequence was actually TCCCACGAC. This meant that the required target sequence at this position was a C, and not a T, as the parts registry web page indicates. The TALE also had a kozak sequence at the front of the sequence, we used PCR to remove the kozak sequence at the front of the TALE, so it would not inhibit the expression
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The central region, also termed repeat region, mediates DNA recognition through tandem repeats of 33 to 35 amino acids residues each (Bogdanove et al., 2010). The binding domain usually comprises 15.5 to 19.5 single repeats (Figure 1). The last repeat, close to the C-terminus, is called “half-repeat” because it is only approximately 20 amino acids in length. Although the modules have conserved sequences, polymorphisms are found in residues 12 and 13, the “repeat-variable di-residue” (RVD). RVDs are specific for a single nucleotide; therefore, 19.5 repeat units target a specific 20-nucleotide sequence in the DNA (Mussolino & Cathomen, 2012).
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In addition to maximizing specificity, we also tried to design both TALEs to have the highest possible binding affinity.
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===Source===
 
===Source===

Revision as of 03:49, 27 September 2013

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


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]


Design Notes

On the iGEM parts registry we found TALB (BBa_K782006) that the Slovenian team synthesized for a previous project. TALEs are very large proteins and take a long time to design and synthesize. Therefore, we decided to use these TALEs to test our system. We also saw this as an opportunity to use and build upon parts made by former iGEM teams. When we sequenced TALB (BBa_K782006), we discovered that it had a small mutated segment. We expected a sequence of AGCAATGGG in the repeat variable di-residue of the second repeat. However, the sequence was actually TCCCACGAC. This meant that the required target sequence at this position was a C, and not a T, as the parts registry web page indicates. The TALE also had a kozak sequence at the front of the sequence, we used PCR to remove the kozak sequence at the front of the TALE, so it would not inhibit the expression

Source

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References