Part:BBa_K3831030:Design
Construct A Construct A for production of BMC
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 250
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 2843
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1108
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
Bidirectional terminator BBa_B0014 is located at the 5' end of composite part A to prevent the activation of our genes from a native promoter in the chromosome of B. subtilis. This terminator is separated from the inducible Pgrac by Spacer_0. Five genes encoding the shell proteins of Pdu BMC from P. thermoglucosidasius are placed downstream of the Pgrac promotor with their corresponding ribosomal binding site in the following order. Translation rate for every sequence and RBS was predicted using an online RBS calculator. RBS_a + PduA (Translation rate = 58 368.33) RBS_b + PduB (Translation rate = 67 646.67) PBS_R2 + PduJ (Translation rate = 57 863.55) RBS_R6 + PduK (Translation rate = 9 221.97) RBS_c + PduN (Translation rate = 5 343.89) Translation rates for PduK and PduN are significantly lower than for the rest of the BMC shell proteins. This should not be an issue as in the Pdu BMCs of S. enterica, which is highly similar to the Pdu BMCs of P. thermoglucosidasius, PduK and PduN are present less than PduA, PduB and PduJ. Forward double terminator BBa_B0015 was placed at the 3' to prevent activation of genes located downstream from the composite part. It was separated from the rest of the sequence by spacer_1 BBa_K3831011. Due to higher degree of similarity in the sequences of the shell proteins of Pdu BMC, this construct could not be synthesized as a whole. It was thus split into two constructs which were then optimised for Golden Gate cloning to allow for them to be ligated together once they have arrived. However, this has caused a bit of a delay. In the end, the synthesized sequences arrived too late and we were unable to proceed with Golden Gate cloning due to time constraints.
Source
Original sequence
References
see all the separate Basic Parts
Guiziou S., Sauveplane V., Chang H. J., Clerté C., Declerck N., Jules M. and Bonnet J. 2016. A part toolbox to tune genetic expression in Bacillus subtilis. Nucleic Acid Res. 44 (15): 7495–7508.
Phan, T. T., Nguyen, H. D., & Schumann, W. (2006). Novel plasmid-based expression vectors for intra- and extracellular production of recombinant proteins in Bacillus subtilis. Protein expression and purification, 46(2), 189–195.
Wade Y., Daniel R. A., Leak D. J. 2019. Heterologous Microcompartment Assembly in Bacillaceae: Establishing the Components Necessary for Scaffold Formation. ACS Synth. Biol. 8: 1642-1654. Kerfeld C. A., Sutter M. 2020. Engineered bacterial microcompartments: apps for programing metabolism. Curr. Opin. Biotechnol. 65: 225-232.