Composite

Part:BBa_K2797006:Design

Designed by: Frank Eardley   Group: iGEM18_Newcastle   (2018-09-28)


Test Device 3 for the iGEM InterLab Study (mNeonGreen)


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 100
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

A simulated Gibson Assembly of the mNeonGreen fluorescent protein sequence into the pSB1C3 vector backbone. As shown, the mNeonGreen slots into the regions in between the biobrick prefix and suffix, a region where the test device is usually present. The green regions show the mNeon green insert. The mNeongreen gene uses the promoter, RBS and terminator of the test device.

Since the idea was to replace the GFPmut3b reporter gene in each of the test device pSB1C3 with the mNeonGreen construct, removal of the GFPmut3b coding region from each vector was required - this was done using 2-step PCR. A reverse primer which binds to the BBa_B0034 RBS and the J23117 promoter of test device 3, and 1 a single forward primer to bind at the beginning of the terminator were utilised in 2-step PCR to linearise the test device 2 pSB1C3 vectors - removing GFPmut3b. The resulting amplification was treated with DpnI.

The mNeonGreen sequence was codon optimised for expression in E. coli DH5-alpha using Benchling. Further to this, Gibson ends were designed using NEBuilder for cloning into the linearised pSB1C3 and added to the 3' and 5' ends of the mNeonGreen sequence. The mNeonGreen sequence was synthesised by IDT. The sequence was cloned into each pSB1C3 vector via Gibson Assembly.

Source

Sequence from Allele Biotechnology

References