Part:BBa_K2052016

FimH site directed mutated with RPMrel and ButCoat

Usage & Biology

This part is composed of a protein coding section (fimH+ RPMrel), a double terminator, arabinose induced promoter, RBS and another protein coding sequence (ButCoaT). FimH protein is a subunit of a structure called pilus, which naturally occurs in some strains of e.coli. This protein coding sequence made our bacteria to have fimH adhesin. Normally, in pathogenic strains of E.coli, at the end of each pilus, there is a carbohydrate binding protein “Lectin” which allows the binding to sugar mannose. However, we used a non- pathogenic strain BL21 in our project so it doesn’t contain Lectin in it’s pili. Thus our bacteria isn’t able to bind to bind to any non-cancerous eukaryotic cells even though it had fimH. Then, to make the binding system cancer specific and to make our bacteria bind only to cancerous cells, we used RPMrel. RPMrel is a 9 amino acid colon tumor specific binding heptapeptide. It was used for controlling preferential binding to poorly-differentiated colon carcinoma cells. (1)

FimH

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 2.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 3. 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.

The part includes a double terminator (BBa_B0015) consisting of BBa_B0010 and BBa_B0012. It works in the forward direction with a forward efficiency of 0.984[CC] and 0.97[JK].

BBa_K206000 is an arabinose induced e.coli promoter. We decided to work with Arabinose Induced Promoter in the middle of our construct to understand if FimH works without putting arabinose and if ButCoaT works with arabinose in the medium.

BBa_B0034 is a ribosome binding site with the efficiency of 1. It is responsible for the recruitment of the ribosomes during the initiation of protein translation.

Co-culture and Fluoresence Microscopy

Here, 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

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.

Butyrate

In order to kill cancerous cells, we decided to overproduce butyrate. Butyrate is a four carbon, short chain fatty acid that inhibits cancer. It induces apoptosis and differentiation, inhibits proliferation of tumorous cells in colon flora. It is produced by the bacterial fermantation of carbohydrates in colon. (2) Butyrate is formed by many pathways which one of them begins with Acetyl-CoA. Acetyl-CoA is readily present in the cells so we wanted to find an enzyme that directly converts it to butyrate, which is ButCoaT. ButCoaT converts acetyl CoA to butyrate by the reaction Butanoyl-CoA + Acetate <=> Butanoic acid + Acetyl-CoA. And BBa_K2052018 is the sequence which codes for ButCoaT.

Figure 1: Butanoyl-CoA + Acetate <=> Butanoic acid + Acetyl-CoA

Figure 2: Crystal Structures of Acetobacter aceti Succinyl (Butyryl)-CoA:Acetate CoA-Transferase Reveal Specificity Determinants and Illustrate the Mechanism Used by Class I CoA-Transferases.(Mullins, E.A. et al., 2012)

RBS and our protein coding region (ButCoaT) are digested from our whole construct with the enzymes SpeI, Sall, Xbal.

1.Ligated Parts Transformation Results

Plate resmi var o eklenecek Resim 8

Figure: After ligating ButcoaT and RFP, we have obtain colonies 2:1 (insert:vector) ratio, and transform them into E.coli BL21.

2.Confirmational PCR Result

resim 9 eklenecek

Figure: Constitutive promoter-RBS-ButCoaT-GFP-Double terminator cloned into pSB1C3 and after ligation we have transformed into E.coli BL21. Colony PCR results here you can see; one primer stick on insert and another stick on vector. The expected lenght of product is around 850 bp.

3.Flowcytometry Result

After checking with confirmational PCR we have tried to validate RFP fluoresence experimentally through Fluorescence Microscope, Here you can see the analysis.

Figure: After measuring RFP signals with channel FL3 we have obtained these graphs. Here again upper triplet stand for Mock(reference point) analysis that is the bacterial culture transformed with only ButCoaT. Middle triplet stand for only RFP expressed bacterial culture analysis which gave a peak that could be used as a control group. The bottom triplet was RFP tagged ButCoaT expressed bacterial group and as we have shown there is any signal depend on RFP activity.

Modeling

Figure 1

In the presence of arabinose The figure above shows the increase in molar concentrations of mRNA of FimH, TetR-LVA, Antiholin in 10 seconds.

Figure 3

The figure above shows the increase in ButCoAT molecules over a time span of 10 seconds.

Figure 4

The figure above shows the molar increase in Butyrate concentration over a time span of 10 seconds.

DNA Gel Analysis

Here, we have uncut and cut version of our whole construct. Since we have out of enzymes, we have digested them with NcoI. After digestion, we were expecting a lane at 1200 bp. Because it has 3 cut sites in the construct, we have extra lanes.

Reference

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

2. Hassig, C.A., Tong, J.K., Schreiber, S.L. (1997). Fiber-derived Butyrate and the Prevention of Colon Cancer