Revision as of 23:32, 19 October 2016

FimH site directed mutated with RPMrel

Usage and Biology

Some substrains of E.Coli have a structure called Type 1 pili which is expressed from the Fim gene system. At the end of the pili structure there is a protein called “FimH” which is the structure that allows them to bind to the mannose sugar that is found on the surfaces of eukaryotic cells. (Sauer et al., 2016).However, the substrain that was used in this project was BL21, a non-pathogenic laboratory strain. Deleted mannose binding and replacing it with RPMrel would provide tumor specific binding (Kelly et al., 2003).The CPIEDRPMC (RPMrel) peptide can bind to five colon cancer cell lines: HT29, CaCo-2, RKO, SW480, and DLD-1. Here on we have choosen CaCo-2 that is studied commonly in METU as our candidate to show targeted thearpy.

3D Structure of FimH

Figure1. 3D Structure of FimH together with RPMrel can be seen below as Harvard BioDesign 2015 submitted in part registry.

Figure2. Our simulations were designed by the NAMD program, and CHARMM force field was applied. This CHARMM technique was used to calculate the energy usage in the simulations. The FimH and HETA models were used in the simulation were taken from an cristal model. After 100 ns, the protein could not disassociate from the ligand. We can understand that there is a strong interaction between the protein and the ligand.

DNA Gel Analysis

Figure 3:In the first 4 lanes the the gene is shown approximately 2100 base pairs and it is uncut. In the firts 4 lanes after the second ladder the gene is shown approximately 3300 base pairs and it is single cut with EcoRI. With the single cut it’s been shown that the insert is successfully in our vector.

Confirmation: PCR

Figure 4: The primers that we have designed bind and multiply the site when the insert and vector are ligated properly, forward binds to a region in vector and reverse binds to a region in insert and give a product at 781 bp for 1,2,3,4 which is K2052014.

Primers: forward 5’-CGAAAAGCCAAAACCTGG-3’-18 bp reverse 5’-GAAGCCTGCATAACGCGG-3’ -18 bp

However , single digestion and conformational PCR has been made to conform that the vector and the insert unites properly. As it can be seen in the first four lanes,PCR gave a hit at 781 bp. It has been conformed that they united well.

Charcterization

Figure 5: The images above represent our co-culture results that we have treated E. coli K12 and E. coli BL21 with CaCo2 cancer cell lines. The pictures show that the cells are sustainable for 6 hours, which is enough for our project. Our observation showed that optimal time for co-culture in our conditions is 6 hours. Then we optimized 1 hour is enough for binding and our treatment trials going to be performed in 5 hours. We expected to see increase in the cell death in a time dependent manner. According to our observation our BL21 forms clusters. However, Harvard BioDesign used E. coli K12 and it was challenging to see them without staining. therefore, BL21 was the best candidate in our case. The protocoles are explained below.

E.Coli BL21 (DE3) & E.Coli K12 MG 1665 Coculture with CaCo-2 Trial Protocol

E.Coli cultures were grown overnight and used in the early stationary phase.

Started from an overnight pre-cultured , stationary phase ( OD > 2) 100 μL sample ( E.Coli : pre-culture ) +10mL LB 37°C , 200 rpm , 16 hours OD600 measurement → OD600 = 1.0 means we have 8 x 10^8 cells 1mL , OD600 = 2.0 → 1,6 x 10^9 cells 1mL ( assumed as stationary phase ) Coculture : CaCo-2 cells were infected with a bacteria at a multiplicity of infection (MOI) of 1000 in 10 mL of cell culture media lacking antibiotics.

This means for 1 cell- 1000 bacteria.

We are growing 3 x 10^4 cell / well, therefore 3 x 10^7 bacteria is required. Overall, 1x 10^9 from each strain is needed.

Determine the amount of well for co-culture. i.e: K12- 25 well x (3 x 10^7 ) = 75 x 10^7 needed

2. Measure OD600 and find out how many cell/mL you have. i.e; We have 12,8 x 10^8 /mL in culture of bacteria.

3. Decide them to find out the amount to centrifuge. i.e; (75 x 10^7) / (12,8 x 10^8 ) =0,585≈0,600

4. Centrifuge (600 µL) from each bacterial culture.

5. Remove flow-through and resuspend in 500 µL medium. (Now, medium is used for cancer cell. However, we could create new media. (mix of EMEM&LB)

6. 500 µL media is needed for each well.Then, 500µL x 25 well= 12,5 mL

7. Prepare media (EMEM) as substracted the amount that we have used to resuspend bacterial strains. So, 12 mL + 500 µL media (resuspended bacteria) =12,5 mL

8. 3hr, 6 hr, 9hr overnight 24 well plates were prepared to carry K12, BL21 and cells only.

9. 12,5 mL of K12, BL21 and EMEM medium were distributed equally and placed into incubator. (No shaking! But maybe we need very slow shaking if we use early passage of CaCo-2.

10. After incubation, plates were washed with PBS as 500 µL for each well. (3 times)

11. 1,25 mL MTT is mixed with 11,25 PBS (total 12,5 mL) and distributed each well as 500 µL after washing.

12. After 4 hours incubation with MTT, add 500 µL SDS (as we have described preparation of SDS) to each well and leave overnight.

Co-culture and Fluoresence Microscopy

Figure 6: Upper left picture shows the cells only and next to it we have taken picture of CaCo2 cells cocultured with Gfp tagged FimH. Bottom pictures were taken under the Fluoresence Microscope but we havent seen any illumination that we have expected. We thought that the fusion of these proteins can change the folding of each protein and resulted in no accumulation of bacteria on cancer cells and any presence of GFP illumination.

• After we havent seen any GFP activity we prepared a new experimental set up for Flowcytometry to be sure whether our control group(only GFP producing bacteria) is working or not and then we wanted to summarize our findings.

Flowcytometry Result

Fluocytometry results fotoğrafları

Figure 7: After measuring GFP signals we have obtained these graphs. Here upper triplet stand for Mock(reference point) analysis that is the bacterial culture transformed with only FimH. Middle triplet stand for only GFP expressed bacterial culture analysis which gave a peak that could be used as a control group. We have obtained the peaks in the FL1 channel of Fluorescence Microscope. The bottom triplet was GFP tagged FimH expressed bacterial group and as we have shown there is any signal depend on GFP activity.

Possible Outcomes of All Our Experimental Findings;

After we did co-culture with CaCo-2 cells, we didn’t observe any clumps around cancer cells, but we observed our freely located bacteria without GFP illumination . We concluded from the results that because there wasn’t any ribosome binding site between GFP and FimH proteins, GFP tagged FimH is unfunctionally formed and the fusion affected their folding

After we did co-culture with CaCo-2 cells, we didn’t observe any clumps around cancer cells, they were freely located but we observed illumination of GFP. We concluded from the results that because there wasn’t any ribosome binding site between GFP and FimH proteins, they fused together which resulted only FimH protein to be affected from this fusion and become unfunctional.

After we did co-culture with CaCo-2 cells, we observed clump formation around only cancer cells but we didn’t observed illumination of GFP. We concluded from the results that because there wasn’t any ribosome binding site between GFP and FimH proteins, they fused together which resulted only GFP protein to be affected from this fusion and become unfunctional.

Modeling

Figure 8: In the presence of arabinose, the figure above shows the increase in molar concentrations of TetR-LVA, Antiholin and mRNA of FimH in a time span of 10 seconds.

Figure 9: In the absence of arabinose, the figure above shows the change in molecule concentrations of Holin mRNA, Holin, Endolysin mRNA, Endolysin, Antiholin and dimer complexes in a time span of 10 seconds.

References

Krogfelt KA., Bergmans H., Klemm P. (1990, June) “Direct evidence that the FimH protein is the mannose-specific adhesin of Escherichia coli type 1 fimbriae”

Sauer MM, Jakob RP, Eras J, Baday S, Eriş D, Navarra G, Bernèche S, Ernst B, Maier T, Glockshuber R. (2016 March 7). “Catch-bond mechanism of the bacterial adhesin FimH”

Kelly, K. A., Jones, D. A., (2003). “Isolation of a Colon Tumor Specific Binding Peptide Using Phage Display Selection”

Figure 1. iGEM 2015 Harvard BioDesign . Retrieved from https://parts.igem.org/Part:BBa_K1850010

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1550331/

Induction protocol http://www.embl.de/pepcore/pepcore_services/protein_expression/ecoli/

Sequence and Features