Difference between revisions of "Part:BBa K4361226"
 
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<partinfo>BBa_K4361226 short</partinfo>
 
<partinfo>BBa_K4361226 short</partinfo>
  
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For the site-directed mutagenesis (SDM) of our protein of interest, BlcR, multiple sets of back-to-back (BTB) primers were designed. The 5' ends of these primers anneal to adjacent nucleotides such that the full plasmid containing the protein ([[Part:BBa_K4361106]]) can be replicated. Each of these numbered sets of primers contains a single reverse primer (denoted as R<i>n</i> with <i>n</i> being the number of the set) and a varying number of forward primers (denoted as F<i>n</i>.<i>m</i> followed by the induced substitution). Every set covers a small range of amino acids in the DNA-binding domain of BlcR, with each position being mutated to different residues by different forward-facing primers. Do note, however, that while a large number of primers have been designed, sequencing showed that the mutagenesis protocol was not successful for all variants. Therefore, only the primers that resulted in the desired mutants will be described in our collection of BioBricks.
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For the site-directed mutagenesis (SDM) of our protein of interest, BlcR, multiple sets of back-to-back (BTB) primers were designed. The 5' ends of these primers anneal to adjacent nucleotides such that the full plasmid containing the protein ([[Part:BBa_K4361106]]) can be replicated. Each of these numbered sets of primers contains a single reverse primer (denoted as R<i>n</i> with <i>n</i> being the number of the set) and a varying number of forward primers (denoted as F<i>n</i>.<i>m</i> followed by the induced substitution). A single amino acid substitution is built-in in the forward primer to introduce a point mutation in the BlcR production plasmid ([[Part:BBa_K4361106]]).
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The mutations are built-in with a site-directed mutagenesis technique. A DNA polymerase is used to lengthen the single-strand primer. After PCR, a Kinase, Ligase, and DpnI (KLD) mix reaction can be performed. The KLD mix contains three enzymes: kinase, for phosphorylation, ligase for ligation, and DpnI for removal of the plasmid template (<b>Figure 1</b>). DNA sequencing is used to identify mutants and determine whether they carry the desired mutation. To know more about the methods we used, see the <html> <a href='https://2022.igem.wiki/tudelft/protocols'>protocols</a> </html> page.
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<a href="https://static.igem.wiki/teams/4361/wiki/design/pcr-and-kld.png"><img src="https://static.igem.wiki/teams/4361/wiki/design/pcr-and-kld.png" style="width:700px;margin-left:75px"></a>
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<figcaption> <b>Figure 1.</b> Site-directed mutagenesis cycle. The first step is the design of the back-to-back primers with the substitution in the forward primer. Second is the amplification of the plasmid with the back-to-back primers to build in the mutation. The third is the phosphorylation by kinase, the ligation by ligase, and the template removal by DpnI.. </figcaption>
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The primer sets are divided as follows:
 
The primer sets are divided as follows:
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     <ul>
 
     <ul>
 
       <li>[[Part:BBa_K4361204]]: R4</li>
 
       <li>[[Part:BBa_K4361204]]: R4</li>
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       <li>[[Part:BBa_K4361205]]: F4.4 A40V</li>
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       <li>[[Part:BBa_K4361205]]: F4.1 A40V</li>
 
     </ul>
 
     </ul>
 
   </li>
 
   </li>
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This primer contains a mutation that changes the codon for valine (V, GTG) in position 68 to that of lysine (K, AAA). <br>
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This primer contains a mutation that changes the codon for valine (V, GTG) in position 68 to that of lysine (K, AAA).<br>
 
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Latest revision as of 15:36, 12 October 2022


BlcR BTB primer F3.8 V68K

For the site-directed mutagenesis (SDM) of our protein of interest, BlcR, multiple sets of back-to-back (BTB) primers were designed. The 5' ends of these primers anneal to adjacent nucleotides such that the full plasmid containing the protein (Part:BBa_K4361106) can be replicated. Each of these numbered sets of primers contains a single reverse primer (denoted as Rn with n being the number of the set) and a varying number of forward primers (denoted as Fn.m followed by the induced substitution). A single amino acid substitution is built-in in the forward primer to introduce a point mutation in the BlcR production plasmid (Part:BBa_K4361106).

The mutations are built-in with a site-directed mutagenesis technique. A DNA polymerase is used to lengthen the single-strand primer. After PCR, a Kinase, Ligase, and DpnI (KLD) mix reaction can be performed. The KLD mix contains three enzymes: kinase, for phosphorylation, ligase for ligation, and DpnI for removal of the plasmid template (Figure 1). DNA sequencing is used to identify mutants and determine whether they carry the desired mutation. To know more about the methods we used, see the protocols page.

Figure 1. Site-directed mutagenesis cycle. The first step is the design of the back-to-back primers with the substitution in the forward primer. Second is the amplification of the plasmid with the back-to-back primers to build in the mutation. The third is the phosphorylation by kinase, the ligation by ligase, and the template removal by DpnI..


The primer sets are divided as follows:

This primer contains a mutation that changes the codon for valine (V, GTG) in position 68 to that of lysine (K, AAA).

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]