Latest revision as of 06:35, 21 October 2016

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.

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

Figure 4: 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.

DNA Gel Analysis

Figure 5: 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.

Confirmation: PCR

Figure 6: 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 lanes stands for K2052016 and 4,5 stands for K2052014(control group).

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.

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

@@ Line 8: / Line 8: @@
 This part is composed of a protein coding section (fimH+ RPMrel), a double
 terminator, arabinose induced promoter, RBS and another protein coding sequence
-(ButCoaT).
+(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==
-FimH protein is a subunit of a structure called pilus, which naturally occurs in some
+===3D Structure of FimH===
-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)
-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
+[[File:METU_HS_2014partinagif.gif|center|frame|100px]]
-Induced Promoter in the middle of our construct to understand if FimH works
+Figure1. 3D Structure of FimH together with RPMrel can be seen below as Harvard BioDesign 2015 submitted  in part registry.
-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.
+[[File:METUITUGf.gif|center|frame|400px]]
+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.
+===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
@@ Line 34: / Line 35: @@
 is the sequence which codes for ButCoaT.
-===Modeling===
+[[File:Pathwayson.png|none|frame|700px|]]
-[[File:MODELING1.jpeg|left|500px]]
+Figure 3: Butanoyl-CoA + Acetate <=> Butanoic acid + Acetyl-CoA
+[[File:ButCoaTStructure.gif|none|frame|500px]]
+Figure 4: 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.
- ‘’’Figure 1 ‘’’
+=DNA Gel Analysis=
-In the presence of arabinose
-The figure above shows the increase in molar concentrations of FimH, TetR-LVA, Antiholin and mRNA in 10 seconds.
+[[File:METU_HS_DENEME77.jpeg|300px]]
-[[File:MODELING2.jpeg|left|500px]]
+Figure 5: 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.
+=Confirmation: PCR=
+[[File:FimH_+_Butcoat.jpg|center|300px]]
+Figure 6: 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 lanes stands for K2052016 and 4,5 stands for K2052014(control group).
+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.
- ‘’’Figure 2 ‘’’
-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 over 10 seconds.
-[[File:MODELING2.jpeg|left|500px]]
- ‘’’Figure3 ‘’’
-The figure above shows the increase in ButCoAT molecules over a time span of 10 seconds.

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