Part:BBa_K4773004:Design
RBS-phi
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
When designing the sequence of this part, we considered the following aspects: - We chose a strong RBS (BBa_B0034) to ensure the efficient expression of the phi gene. - We chose a phi gene from a methylotrophic bacterium, to ensure the high activity and stability of PHI enzyme. - We added a KpnI restriction site after the rsrr sequence, to facilitate its connection with other parts. KpnI is a restriction enzyme that can recognize and cut the GGTACC sequence. We avoided this sequence in the rsrr or phi gene, to prevent it from being wrongly cut. - We did not add a terminator at the end of the phi 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 phi gene. The RBS can enhance the transcription and translation of the phi gene, increasing the expression level of PHI enzyme. The phi gene originates from a methylotrophic bacterium Mycobacterium gastri MB19, which has a high PHI activity and stability. The PHI enzyme can work at room temperature and neutral pH, catalyzing the reversible reaction between Hu6P and F6P. This part can be expressed in different host cells, enabling them to utilize or detoxify formaldehyde.