Our part collection offers a complete array of parts for future researchers to develop other probiotic secreted proteins/peptides.
Updated on 2020-11-22: we were nominated for the Best Part Collection prize.
In the collection, we have secreted mcbABCD to help its colonization in the gut, and secreted CaAP to facilitate calcium uptake by gut cells. We test all parts used in these two circles: 5 signal peptide for secretion, 14 promoters for different strength expression (previously publish and available in addgene.org but not in the Registry), IPTG inducible promoter driven various protein coding sequences (confirmed individually before assembled together), and quorum-sensing components. A file contains those parts could be downloaded (updated on 2020-10-27).
We not only provide sequences and references for the parts, but also experimental results and procedures /Team:Fudan/Experiments. We compete for both the Gold Medal criterion #7 and the Best Part Collection prize with this page.
Five signal peptide for secretion
We have constructed plasmids using CaAP or MBP (Maltose Binding Protein, a protein that is expressed in the cytoplasm of E. coli) with five signal peptides (CaAP fused with signal peptides: BBa_K3606031~BBa_K3606035; MBP fused: BBa_K3606037~BBa_K3606041). We have quickly test several of them: the total production and relative amount being secreted. We show that OmpA, one of our signal peptides, could lead MBP to secret. Our results consist with the literature that for a specfic protein all possible signal peptides should be test for best results.
Figure 1. TCA precipated culture media (lane 8/with 9/no IPTG OmpA-MBP)
Lane 8 and 9, bacteria DH5a with OmpA-MBP, culture to OD 0.6 and induced with 1mM IPTG for 6 hours. Then, the culture media was collected and precipated with TCA. Samples were run on 17% SDS-PAGE. Lane 1, aprotinin 6.5 kDa; Lane 10, pMAL-c2g induced with same IPTG to show the size of cytoplasmic MBP.
Figure 2. IPTG induced signal peptide fused CaAP
When wiki freeze, we do NOT have Plac driven PhoA-CaAP.
Bacteria DH5a w/ pGEX-6P1, culture to OD 0.6; (lane 5) was induced with 1mM IPTG for 6 hours. Then, the bacteria whole-cell samples were run on 17% SDS-PAGE. Lane 1, aprotinin 6.5 kDa.
14 promoters for different strength expression
Figure 3. Bacteria with P1-P14 driven RFP incubated in 3mL medium for 12h
Figure 4. Bacteria with P1-P14 driven RFP incubated in 3mL medium for 24h
Figure 5. Ni-NTA bound CaAP
Red dot, aprotinin 6.5 kDa, 16% SDS-PAGE. Lane 1, P3 driven CaAP; Lane 2, P4 driven CaAP; Lane 3, P5 driven CaAP; Lane 4, P6 driven CaAP; Lane 5, P7 driven CaAP; Lane 6, P8 driven CaAP; Lane 7, pGEX-6P1 (no 6xHis); Lane 8, P11 driven CaAP; Lane 9, P12 driven CaAP. For each sample, 3 OD (2 ml) bacteria were sonicated to lysis. And, 50 uL Ni-NTA beads (50% slurry) were mixed with cleared lysate for binding. Beads were washed in Buffer A, and then half of the beads were boiled in 100 uL with 1x SDS sample buffer, 10 uL was loaded to the lane.
Using obtained P1-P14 driven RFP, we have constructed constructs expressing CaAP with 14 promoters for different strength expression (BBa_K3606045~BBa_K3606057). Those bacteria were grew to OD 1.5, broken up, and Ni-NTA beads were added into cleared lysate. A quick pull-down show clear bands of protein on the gel, which were bound to Ni-NTA beads, very likely are CaAP!
IPTG inducible promoter driven mcbABCDEFG
First, to affirm the system could work properly, we started from the very beginning by testing the expression of each gene in the cluster as well as of the polycistrons in each part. We constructed 10 plasmids (with pGEX backbone adapted to perform high level of expression), containing mcbA/ mcbB/ mcbC/ mcbD/ mcbE/ mcbF/ mcbG/ mcbABCD/ mcbBCD/ mcbBC respectively, transformed them into E. coli BL21, then induced with IPTG. We confirmed that every part can express successfully due to the correct bands on the SDS-PAGE electrophoresis gel maps.
Figure 6. IPTG induced mcbE mcbF
Red dot, aprotinin 6.5 kDa; lane 2 vs 1, mcbE induced by IPTG; lane 4 vs 3, mcbF induced by IPTG; lane 6 vs 5, GST induced by IPTG. Bacteria were grown to OD 0.6, induced with or without 1 mM IPTG for 5 hours. Whole-cell samples were run. Yellow dots were placed to show induced bands in lane 2,4,6.
When wiki freeze, we do NOT have Plac driven mcbG.
2020 Team Fudan parts at parts.igem.org, including:
Signature: Yuanyuan