Difference between revisions of "Part:BBa K2052015"
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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 : | 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 : | ||
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[[File:Pathwayson.png|none|frame|700px|]] | [[File:Pathwayson.png|none|frame|700px|]] | ||
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Figure 1: Butanoyl-CoA + Acetate <=> Butanoic acid + Acetyl-CoA | Figure 1: Butanoyl-CoA + Acetate <=> Butanoic acid + Acetyl-CoA | ||
| − | [[File:ButCoaTStructure.gif|none|frame|500px]] | + | [[File:ButCoaTStructure.gif|center|none|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) | 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. | RBS and our protein coding region (ButCoaT) are digested from our whole construct with the enzymes SpeI, Sall, Xbal. | ||
| − | + | ==Cloning== | |
| − | ==Gel Results== | + | ====Gel Results==== |
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| + | [[File:METU_HS_DENEME8.jpeg|center|400px]] | ||
| + | Figure 3. We have loaded an uncut version of K2052015 next to an EcoRI single digested one. In the third lane RBS ligated ButCoat (K2052018) was loaded with its single digested version. Digested ones gave a sharp lane at 3600 bp as we expected. | ||
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==Characterization== | ==Characterization== | ||
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| − | ==Ligated Parts Transformation Results== | + | ===Ligated Parts Transformation Results=== |
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| − | + | [[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. | ||
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| + | ===Confirmational PCR Result=== | ||
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| − | + | [[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. | ||
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| + | ==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. | |
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| − | + | 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. | |
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| − | + | ==Modelling== | |
| − | + | Molecule versus second | |
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| − | + | [[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 | |
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| + | [[File:Butyrate graph.jpeg|900px]] | ||
| + | Figure 8.The figure above shows the molar increase in Butyrate concentration over a time span of 10 seconds. | ||
Latest revision as of 00:32, 20 October 2016
ButCoat with strong RBS
Usage & Biology ( Description )
This composite part consists of RBS , 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)
RBS and our protein coding region (ButCoaT) are digested from our whole construct with the enzymes SpeI, Sall, Xbal.
Cloning
Gel Results
Figure 3. We have loaded an uncut version of K2052015 next to an EcoRI single digested one. In the third lane RBS ligated 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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]