Difference between revisions of "Team:CAU China/Parts"

    In the cellulose degradation subsection, we created a part INP-N(BBa_K3279006), which is the N terminus of Ice-nucleation protein from Pseudomonas syringae, and we fused it with the existing parts CenA(BBa_K118023) and Cex(BBa_K118022) to achieve the goal of surface display of cellulases. The sequence encoding the fused protein INPN-CenA(BBa_K3279008) is created as an improved part of CenA. We also characterized the part Cex and measured its ability to degrade cellulose by the activity assay.

    Ice-nucleation protein (INP) is a secreted outer membrane protein, which is widely distributed in Pseudomonas syringae, Pseudomonas fluorescens and other Gram-negative bacteria. Compared with other surface carrier proteins, ice-nucleation protein has the advantage of stably expressing heterogeneous proteins and displaying proteins with larger molecular weight. This part includes the sequence of INP-N (N terminus of Ice-nucleation protein), which is much shorter than INP and INP-NC yet still functions to anchor the target protein on the cell outer membrane. To allow this part fusion with other protein, we added an additional linker sequence at the 3'.

    We linked the sequence of Cex gene (BBa_K118022) into the pET30a(+) backbone, which contains lacI sequence so that the heterogeneous proteins can be induced by IPTG. The plasmids were transferred into BL21(DE3) strain and we induced the recombinant overnight under the condition of 16℃ 0.08 mM IPTG. The expression of the fusion protein was determined by SDS-PAGE.

    The activity of Cex was determined by measuring the cellulose degradation ability using CMC-Na as the substrate. We employed the partial procedures used by UESTC-China yet under the condition of citric acid-sodium citrate buffer with pH 4.8 and 50 ℃ for reaction temperature. The cells were disrupted by ultrasonication and the suspension of the centrifugal cell contents was used as the crude enzyme. We measured the cellulose degradation abilities of the crude enzyme.

    We improved the part encoding Cellulomonas fimi endoglucanase A (CenA, BBa_K118023) by fusing it with INP-N (BBa_K3279006) and created a new part INPN-CenA (BBa_K3279008), INPN-CenA can anchor in the cell outer membrane and function for surface display. The fusion protein was expressed and confirmed by SDS-PAGE, the anchoring effect is determined through immunofluorescence staining and enzyme activity assay.

    In the astaxanthin synthesis subsection, we created three parts: Geranylgeranyl diphosphate synthase (CrtE) from Rhodobacter sphaeroides (BBa_K3279000), Phytoene Synthase (CrtB) from Rhodobacter sphaeroides (BBa_K3279001) and Phytoene desaturase (CrtI) from Rhodospirillum rubrum (BBa_K3279002). All of the three parts are crucial enzymes in astaxanthin synthesis pathway. We characterized the latter two parts quantitively but remained the first part only sequence information.

    There was an illegal restriction enzyme site in the original sequence of CrtE, so we made a site-specific mutation to remove it. The mutation results was shown on the main page of BBa_K3279000.

    The CrtB and CrtI were characterized together, but we are convinced that both of the two parts worked. Because the two genes are upstream and downstream, like two components in a series circuit. If either of them broke down, the engineered E.coli cells could not produce lycopene. We tested the ability of these two genes to produce lycopene at different IPTG concentrations and different induction times. The detailed experiment results was shown on the Results Page.