Part:BBa_K4451021:Design
MutaT7 Test Cassette ACG
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 1599
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal PstI site found at 1599 - 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 850
Illegal XhoI site found at 1731
Illegal XhoI site found at 1937 - 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 1599
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 1599
Illegal AgeI site found at 561 - 1000COMPATIBLE WITH RFC[1000]
Design Notes
All protein-coding sequences within BBa_K4451021 have been codon-optimised for expression in Vibrio natriegens, since the eventual goal of the Sheffield 2022 project was to engineer a plasmid-based continuous in vivo directed evolution system using a V. natriegens chassis.
BBa_K4451021 was created from two 1kbp IDT-synthesised gBlocks, then assembled into a pBBRBB derived plasmid backbone using NEB HiFi DNA assembly. Verified E. coli DH5α transformants were streaked onto streptomycin and chloramphenicol plates, resulting in no growth and thereby suggesting that ACA cannot function as an alternative start codon.
Source
References
Mairhofer, J., Wittwer, A., Cserjan-Puschmann, M. & Striedner, G. Preventing T7 RNA Polymerase Read-through Transcription—A Synthetic Termination Signal Capable of Improving Bioprocess Stability. ACS Synth. Biol. 4, 265–273 (2015).
Moore, C. L., Papa, L. J. & Shoulders, M. D. A Processive Protein Chimera Introduces Mutations across Defined DNA Regions In Vivo. J. Am. Chem. Soc. 140, 11560–11564 (2018).