Part:BBa_K4773003:Design
RBS-hps
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 367
Design Notes
We chose a strong RBS (BBa_B0034) to ensure the efficient expression of the hps gene. We chose an hps gene from a methylotrophic bacterium to ensure the high activity and stability of the HPS enzyme. We added restriction enzyme sites at both ends of the hps gene, to facilitate its connection with other parts. We used BpiI and BsaI, two common restriction enzymes, that can recognize and cut specific sequences . We avoided these sequences in the hps gene, to prevent it from being wrongly cut. We did not add a terminator at the end of the hps gene, because we wanted to splice it with other genes into a multifunctional fusion protein. If we added a terminator, it would block the expression of downstream genes. We also considered some factors that affect prokaryotic expression, such as codon bias, RNA secondary structure, protein folding, etc. We tried to choose codons that match the host bacteria, avoid forming stable RNA secondary structures, and increase protein folding helper factors.
Source
This part consists of a strong bacterial ribosome binding site (RBS) (BBa_B0034) and the hps gene. The RBS can enhance the transcription and translation of the hps gene, increasing the expression level of HPS enzyme. The hps gene originates from a methylotrophic bacterium Mycobacterium gastri MB191, which has a high HPS activity and stability. The HPS enzyme can work at room temperature and neutral pH, catalyzing the reversible reaction between formaldehyde and Ru5P. This part can be expressed in different host cells, enabling them to utilize or detoxify formaldehyde.