This year, we have summited five new parts to the registry to prove our Engineering Success.
To make engineering bacteria obtain the capacity of adhesion, we decid to insert hsa from Streptococcus gordonii str. Challis into pET-28a(+) and transform the plasmid into E. coli BL21(DE3) by infusion cloning. Hsa can anchor to the cell wall and bind to sialic acid in the cat's mouth, helping our bacteria to adhere to the salivary pellicle. The hsa has a length of 6537 bp. To reduce the weight of the vector, we only selected the regions in hsa that can bind to sialic acid. After transforming pET-28a(+) with hsa into E. coli BL21(DE3), we intend to perform functional validation in terms of both biofilm formation ability and adhesion to saliva-coated cat tooth.
Figure 1.Gel electrophoretic map after transforming the constructed plasmid into E. coli BL21 (DE3).
But after further literature review, we found that hsa is divided into five regions, NR1, SR1, NR2, SR2, and CWAD, with NR2 being the main salivary acid-binding site, SR2 acting as a molecular stalk and CWAD is the cell wall anchoring region. We only intercepted a portion of NR2, and don't need experimental verification to know that our genes cannot function in bacteria. So after literature references, we chosing csgA associated with biofilm from E. coli K-12. CsgA is a Curli amyloid fibrils protein secreted extracellularly to mediate host cell adhesion and contributes to biofilm formation. We intercepted the NR2 region of Hsa and linked it to the C-terminus of CsgA with the linker of (GGGGS)4, and linked the fusion gene to pET-28a(+) by homologous recombination, named csgA-hsa. And by SDS-PAGE analysis we identified the expression of CsgA-Hsa.
Figure 2. Gel electrophoretic map after transforming the pET-28a(+) with csgA-hsa into E.coli BL21(DE3).
Figure 3. SDS-PAGE analysis of CsgA-Hsa.
After completing the vector construction and protein expression validation of CsgA-Hsa, we began adhesion ability on cat tooth and quantitative and qualitative of biofilm experiments.
We covered the induced bacterial debris on the saliva coated cat tooth, and after elution and antibody incubation, there was a significant difference in fluorescence intensity between the experimental group and the control group, fully demonstrating the ability of CsgA-Hsa to bind to sialic acid.
Figure 4. The results ofadhesion ability on cat tooth.
Incubating the induced engineering bacteria in a 96 well plate, giving them time to form a biofilm, then stain with crystal violeted and detected OD600 values using an enzyme-linked immunosorbent assay. The significant difference in results between the experimental group and the control group indicated that our engineered bacteria have formed a biofilm.
Figure 5. OD600 values after crystal violet staining.
Cultivating and inducing engineered bacteria in a 6-well plate at 37°C, then eluting, fixing, and staining them. We observed the result with a fluorescence microscope after staining engineered bacteria. Under the induction of 0.1mM IPTG, the amount of biofilm formation was the highest and far exceeded that of other concentration groups and control groups.
Figure 6. Fluorescence images of gradient-induced bacterial biofilm.
For more details, please click the link below:http://parts.igem.org/Part:BBa_K4645003
NeuA and NeuB are scFvs that we use rigid linker to fuse the light chain and heavy chain, and they have the ability to bind to Fel d 1 and prevent IgE from people who are allergic to cats engagement.
Figure 7. SDS-PAGE analysis of purifed FelD, NeuA and NeuB.
Figure 8. OD450-Concentration curve of block ability of FelD to IgE from allergic human donor sera or Sstandard IgE sample.
To verify the blocking activity of our scFvs, we first expressed recombinant Fel d 1, FelD (Figure 7, A). The ability to bind to allergic IgE was also verified (Figure 8). Subsequently, we successfully expressed and purified NeuA and NeuB (Figure 7, B), and verified by blocking ELISA that they have blocking activity and better blocking ability when used together (Figure 9).
Figure 9. The result of blocking ELISA. Values represent the effect of binding allergen and preventing IgE engagement.
For more details, please click the link below: BBa K4645000, BBa K4645001,BBa_K4645002
At first, we intented to use VqmA and Pqtip to sense the concentration of bacteria. By SDS-PAGE analysis we identified the expression of VqmAphage.
Figure 10. SDS-PAGE analysis of VqmAphage
However, Pqtip didn't work. We found that the sequence of qtip promoter contains more than one initiation codon. This may lead to the losing of efficiency of ECFP. So, we tried to delete sequence between eCFP and the last initiation codon and between eCFP and the penult one. Sad to say, we were not able to make this part work finally.
So, we use AHL quorum sensing system to regulate the circuit. 50 μL E. coli BL21(DE3) with 50μL luxR (BBa_K4115039) + 50 μL E. coli BL21(DE3) and 50 μL luxR (BBa_K4115039) + 50 μL E. coli BL21(DE3) with luxI (BBa_C0161) was respectively mixed with 5 mL LB medium. Then they were cultured in microplate reader 37°C, 220 rpm for 8 hours and detected OD600, fluorescence intensity (458 nm excitation light, 489 nm emission light) every 10 minutes.
Figure 11. Fluorescence intensity in different value of OD600.
Later, we built a circuit (BBa_K4645019) to test whether Not-Gate could workor not. Blank E.coli BL21(DE3), engineered E.coli BL21(DE3) with this biobrick were cultured in microplate reader 37°C, 220 rpm for 10 hours and detected OD600, fluorescence intensity (458 nm excitation light, 489 nm emission light) every 10 minutes.
Figure 12. Changes of fluorescence intensity / OD600 in blank E.coli BL21(DE3), engineered E.coli BL21(DE3) with this biobrick over time.
The result shows that in the early growth stage, transformed bacteria expressed amcyan fluorescent protein continually. When the group density reached the threshold value, the expression of amcyan fluorescent protein began to be blocked up.
For more details, please click the link below: BBa_K4645004, BBa_K4645005, BBa_K4645016, BBa_K4645019