Difference between revisions of "Part:BBa K2052018"

Latest revision as of 01:17, 20 October 2016

Butyryl-CoA:acetate CoA-transferase

Usage & Biology

This basic part consists of a coding region ( ButCoAT ) and enables E.coli to secrete ButCoAT.

ButCoAT is an enzyme (Butyryl-CoA:acetate CoA-transferase) (EC 2.8.3.8) (Uniprot:C7GB37) which converts Acetyl-CoA (Acetyl-CoA is readily present in the cytosol and it can be directly converted into Butyrate) into Butyrate and can be found in Roseburia intestinalis L1-82. The equation of the reaction (acyl-CoA:acetate CoA-transferase) is given down below :

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)

Our protein coding region (ButCoaT)is digested from our whole construct with the enzymes PstI and Xbal.

Gel Results

We have loaded an uncut version of K2052015 next to an EcoRI single digested one. In the third lane ButCoat (K2052018) was loaded with its single digested version. Digested ones gave a sharp lane at 3600 bp as we expected.

Characterization

Ligated Parts Transformation Results

Figure 4. After ligating ButcoaT and RFP, we have obtain colonies 2:1 (insert:vector) ratio, and transform them into E.coli BL21.

Confirmational PCR Result

Figure 5. 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.

Flowcytometry Result

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

Figure 6. 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.

Since ButCoAT was tagged with RFP, to test whether it is functional or not, we have used fluoresence microscopy technique to show its expression is properly done or not. However, because ButCoAT doesn’t have a stop codon at the end it fused with RFP and they both become unfunctional from this fusion. Therefore, we could not see any peak.

Modelling

Molecule versus second

Figure 7.The figure above shows the increase in ButCoAT molecules over a time span of 10 seconds.

Molecule versus second

Figure 8.The figure above shows the molar increase in Butyrate concentration over a time span of 10 seconds.

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K2052018 short</partinfo>
-.............
-Enter a long description of the part so that users of your part know what it is, what it does, and how to use it in their projects.
-<!-- Add more about the biology of this part here
+==Usage & Biology==
-===Usage and Biology===
-===Butyrate===
-Butyrate is a four carbon, short chain fatty acid that inhibits cancer. It induces
+This basic part consists of a coding region ( ButCoAT ) and enables  E.coli to secrete ButCoAT.
-apoptosis and differentiation, inhibits proliferation of tumorous cells in colon flora. It is produced by the bacterial fermantation of carbohydrates in colon. Butyrate is formed by many pathways that begin with glutarate, Acetyl-CoA and Lysine as it is seen in the picture below. Acetyl-CoA is readily present in the cells so we want to find an enzyme that directly converts it to our treatment, Butyrate which is ButCoAT
+ButCoAT is an enzyme (Butyryl-CoA:acetate CoA-transferase) (EC 2.8.3.8) (Uniprot:C7GB37) which converts Acetyl-CoA (Acetyl-CoA is readily present in the cytosol and it can be directly converted into Butyrate) into Butyrate and can be found in Roseburia intestinalis L1-82. The equation of the reaction (acyl-CoA:acetate CoA-transferase) is given down below :
-<!-- -->
+[[File:Pathwayson.png|none|frame|700px|]]
-<span class='h3bb'>Sequence and Features</span>
-<partinfo>BBa_K2052018 SequenceAndFeatures</partinfo>
+Figure 1: Butanoyl-CoA + Acetate <=> Butanoic acid + Acetyl-CoA
-<!-- Uncomment this to enable Functional Parameter display
-===Functional Parameters===
-<partinfo>BBa_K2052018 parameters</partinfo>
+[[File:ButCoaTStructure.gif|none|center|frame|500px]]
-<!-- -->
+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)
+Our protein coding region (ButCoaT)is digested from our whole construct with the enzymes PstI and Xbal.
+===Gel Results===
+[[File:METU HS DENEME688889.jpeg|500px]]
+We have loaded an uncut version of K2052015 next to an EcoRI single digested one. In the third lane ButCoat (K2052018) was loaded with its single digested version. Digested ones gave a sharp lane at 3600 bp as we expected.
+==Characterization==
+===Ligated Parts Transformation Results===
+[[File:METU HS DENEME9.jpeg|center|250px]]
+Figure 4. After ligating ButcoaT and RFP, we have  obtain colonies 2:1 (insert:vector) ratio, and transform them into E.coli BL21.
+===Confirmational PCR Result===
+[[File:METU_HS_DENEME6888.jpeg|center|500px]]
+Figure 5. 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.
+==Flowcytometry Result==
+After checking with confirmational PCR we have tried to validate RFP fluoresence experimenttaly through Fluorescence Microscope, Here you can see the analysis.
+[[File:METU_HS_DENEME3luolan.jpeg|center|800px]]
+Figure 6. 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.
+Since ButCoAT was tagged with RFP, to test whether it is functional or not, we have used fluoresence microscopy technique to show its expression is properly done or not. However, because ButCoAT doesn’t have a stop codon at the end it fused  with RFP and they both become unfunctional from this fusion. Therefore,  we could not see any peak.
+==Modelling==
+Molecule versus second
+[[File:ButCoAT_graph.jpeg|900px]]
+Figure 7.The figure above shows the increase in ButCoAT molecules over a time span of 10 seconds.
+Molecule versus second
+[[File:Butyrate graph.jpeg|900px]]
+Figure 8.The figure above shows the molar increase in Butyrate concentration over a time span of 10 seconds.