Difference between revisions of "Part:BBa K2052015"

Revision as of 00:28, 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 1. 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: 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: 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: 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

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

Molecule versus second

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

Sequence and Features