Difference between revisions of "Part:BBa K2271107:Design"
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===Part design===
 
===Part design===
 
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   This part was designed in the course of a random mutagenesis approach. With the predicted three dimensional strucutre of the TPR domains we carried out our workflow in order to obtain the PEX5 variant we wanted.
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   This part was designed in the course of a targeted mutagenesis approach. For this Pex5 variant we considered recently published literature (Towards designer organelles by subverting the peroxisomal import pathway - Baker <i>et al.</i>, 2017) and designed a receptor that should interact with a PTS1 variant. We performed molecular dynamics simulations to test how benefitial the mutations were and synthesized the Pex5 gene by IDT.
 
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   After gene synthesis we performed golden gate cloning and made it compatible to the biobrick standard via overhang PCR with the appropriate primers. This product then was ligated into the biobrick backone <b>pSB1C3</b>.
 
   After gene synthesis we performed golden gate cloning and made it compatible to the biobrick standard via overhang PCR with the appropriate primers. This product then was ligated into the biobrick backone <b>pSB1C3</b>.
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   The original sequence was taken from NCBI (GenBank: KZV12481.1). After mutagenesis we performed the IDT codon optimization for <i>Saccharomyces cerevisiae</i>, removed any restrictions sites that would interfere with the biobrick or golden gate standard and synthesized the gene with appropiate Golden Gate overhangs.
 
   The original sequence was taken from NCBI (GenBank: KZV12481.1). After mutagenesis we performed the IDT codon optimization for <i>Saccharomyces cerevisiae</i>, removed any restrictions sites that would interfere with the biobrick or golden gate standard and synthesized the gene with appropiate Golden Gate overhangs.
 
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===References===
 

Revision as of 18:53, 1 November 2017


PEX5 variant R19 with Promotor/Terminator


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1341
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1128
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1633
  • 1000
    COMPATIBLE WITH RFC[1000]


Part design

This part was designed in the course of a targeted mutagenesis approach. For this Pex5 variant we considered recently published literature (Towards designer organelles by subverting the peroxisomal import pathway - Baker et al., 2017) and designed a receptor that should interact with a PTS1 variant. We performed molecular dynamics simulations to test how benefitial the mutations were and synthesized the Pex5 gene by IDT.
After gene synthesis we performed golden gate cloning and made it compatible to the biobrick standard via overhang PCR with the appropriate primers. This product then was ligated into the biobrick backone pSB1C3.

Source

The original sequence was taken from NCBI (GenBank: KZV12481.1). After mutagenesis we performed the IDT codon optimization for Saccharomyces cerevisiae, removed any restrictions sites that would interfere with the biobrick or golden gate standard and synthesized the gene with appropiate Golden Gate overhangs.