Our part collection offers a complete array of parts for further researchers to develop other probiotic secreted proteins/peptides.
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.
Click here to see more detailsWe not only provide sequences and references for the parts, but also experimental results and procedures. We compete for both the Gold Medal criterion #7 and the Best Part Collection prize with this page.
1. 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 (BBa_K3606031~BBa_K3606035). We plan to test their amount of secretion and production in the same period. We have proven that OmpA, one of our signal peptides, can guide MBP to secret. We hypothesis the difference between each signal peptide is significant.
2. 14 promoters for different strength expression
Figure 1. Measurement of bacteria incubated in 3mL medium for 12h
Figure 2. Measurement of bacteria incubated in 3mL medium for 24h
We have constructed plasmids using RFP with 14 promoters for different strength expression (BBa_K3606045~BBa_K3606057) and have tested their fluorescence intensity. From the form above, the difference between each promoter is significant.
3. 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 purified the protein products with his-tag via immobilized metal affinity chromatography to check if the basic protein-coding could all be realized correctly as the basis of our design for the system. We confirmed that every part can express successfully due to the correct bands on the SDS-PAGE electrophoresis gel maps.