Difference between revisions of "Part:BBa K2797003:Design"

 
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===Design Notes===
 
===Design Notes===
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Codon optimisation for expression in E. coli
 
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An mNeonGreen construct was designed for use as an alternate fluorescent reporter for each test device - replacing GFP. The mNeonGreen sequence was codon optimised using Benchling and Gibson ends were designed using NEBuilder for cloning into pSB1C3. The subsequent sequence was synthesised by IDT. Prior to the PCR linearisation, pSB1C3 plasmid concentration for each mini-prepped test device was determined using a Qubit fluorometer and diluted to 0.5 ng/µl. The diluted pSB1C3 vectors were linearised using a 2 step PCR system following a Q5 Polymerase protocol (NEB). This protocol utilised forward and reverse primers with Tm values of 72°C. The mNeonGreen primers were designed by using the NEB Tm calculator and Benchling. The amplified DNA was then digested with DpnI, heat treated to inactivate the enzyme and assembled via Gibson Assembly using the NEBuilder HiFi DNA Assembly Kit. Following their protocol, a 2-fragment reaction with 0.5 pmol of DNA in a 2:1 insert to vector ratio was done and transformants were plated onto agar plates with the appropriate antibiotic (LB+cam for each test device and LB+amp for the controls). Following growth of colonies, plasmid DNA was miniprepped from DH5-α transformed with both the internal standard and the mNeonGreen vector and sequenced to verify presence of the genes.
  
 
===Source===
 
===Source===

Revision as of 16:34, 2 October 2018


mNeonGreen


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 39
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

An mNeonGreen construct was designed for use as an alternate fluorescent reporter for each test device - replacing GFP. The mNeonGreen sequence was codon optimised using Benchling and Gibson ends were designed using NEBuilder for cloning into pSB1C3. The subsequent sequence was synthesised by IDT. Prior to the PCR linearisation, pSB1C3 plasmid concentration for each mini-prepped test device was determined using a Qubit fluorometer and diluted to 0.5 ng/µl. The diluted pSB1C3 vectors were linearised using a 2 step PCR system following a Q5 Polymerase protocol (NEB). This protocol utilised forward and reverse primers with Tm values of 72°C. The mNeonGreen primers were designed by using the NEB Tm calculator and Benchling. The amplified DNA was then digested with DpnI, heat treated to inactivate the enzyme and assembled via Gibson Assembly using the NEBuilder HiFi DNA Assembly Kit. Following their protocol, a 2-fragment reaction with 0.5 pmol of DNA in a 2:1 insert to vector ratio was done and transformants were plated onto agar plates with the appropriate antibiotic (LB+cam for each test device and LB+amp for the controls). Following growth of colonies, plasmid DNA was miniprepped from DH5-α transformed with both the internal standard and the mNeonGreen vector and sequenced to verify presence of the genes.

Source

Allele Biotechnology

References