Team:IISER-Bhopal-India/Improve
Parts Submitted
BBa_K2790004 (P2A1)
This composite part can be used for the expression of three proteins (sMMO C- 1047bp, sMMO D- 312bp and sMMO Y- 1170bp) from the methanotrophic bacteria M. capsulatus. The sequences of these 3 proteins are codon optimised for expression in the methylotrophic yeast P. pastoris. These three proteins are subunits of the enzyme methane monooxygenase which converts methane into methanol. The expression is driven by the strong constitutive GAP promoter from P. pastoris and ends with the AOX terminator from P. pastoris. The three genes are linked together with viral peptide P2A sequences and hence, their mRNA will be transcribed as a whole but during translation, they will form individual polypeptides due to ribosome skipping a peptide bond between the proline and glycine residue at the C-terminus of the viral 2A peptide. This construct drastically reduces the expression cassette size since each of the genes need not be flanked by their own promoter and terminator rather can be controlled by a single promoter similar to a prokaryotic operon. Also, the order of the genes in the construct is insignificant since the 2A peptides would give a similar expression for the three proteins. In total, this part provides an expression casette for the constitutive expression of three sMMO subunits codon optimised for P. pastoris but can also be expressed in S. cereviseae.
BBa_K2790005 (P2A2)
This composite part can be used for the expression of three proteins (sMMO B- 426bp, sMMO X- 1584bp and sMMO Z- 510bp) from the methanotrophic bacteria M. capsulatus. The sequences of these 3 proteins are codon-optimized for expression in the methylotrophic yeast P. pastoris. These three proteins are subunits of the enzyme methane monooxygenase which converts methane into methanol. The expression is driven by the strong constitutive GAP promoter from P. pastoris and ends with the AOX terminator from P. pastoris. The three genes are linked together with viral peptide P2A sequences and hence, their mRNA will be transcribed as a whole but during translation, they will form individual polypeptides due to ribosome skipping a peptide bond between the proline and glycine residue at the C-terminus of the viral 2A peptide. This construct drastically reduces the expression cassette size since each of the genes need not be flanked by its own promoter and terminator but can be controlled by a single promoter similar to a prokaryotic operon. Also, the order of the genes in the construct does not matter since the 2A peptides would give a similar expression for the three proteins. In total, this part provides an expression cassette for the constitutive expression of three sMMO subunits codon optimized for P. pastoris but can also be expressed in S. cereviseae.
Improved Parts
BBa_K2790006 (sMMO D-AoxPromoter-iLOV)
The submitted part Bba_K2790006 is a 3A assembly of smmoD gene flanked by a eukaryotic promoter and a promoter-reporter (AOX promoter-iLOV) construct. Amplification of the latter can be done by specific primers for AOX promoter-iLOV construct and the amplicon can be cloned into desired vectors. The AOX-iLOV is an improvement over the AOX promoter-RFP construct as iLOV is a better fluorescence protein than RFP or GFP as shown already in characterization by Team Exeter in 2014. The key points which make AOX promoter-iLOV an improvement over AOX promoter-RFP are: