Part:BBa_K2052015
ButCoat with strong RBS
Usage & Biology ( Description )
This composite part consists of RBS , our 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 to Butyrate and can be found in Roseburia intestinalis L1-82.
The equation of the reaction (acyl-CoA:acetate CoA-transferase) (KEGG Reaction No. : R01179) is given down below : Butanoyl-CoA + Acetate <=> Butanoic acid + Acetyl-CoA
RBS and our protein coding region (ButCoaT) are digested from our whole construct with the enzymes SpeI, Sall, Xbal.
For ButCoaT ;
E.Coli is synthesised suitable for Codon Bias. In the expression of pet28a (expression vector) , stop codon is removed in order to add HisTag.
Characterization
After growing and obtaining colonies in Overnight culture, we have got optical density at 1.0 and by using several techniques we have extracted and loaded proteins on a SDS gel. Without induction, the constitutive promoter that we have ligated the previous year worked perfectly and gave us a lane at 38kDA. The control group was the e. coli dh5a untransformed colonies. Here you can see the image;
First well is ladder. 3rd lane of ladder from bottom stands for 25. 4th lane from bottom stands for 55kDa. First well is loaded with empty dh5a and its lysate is just right next to it 3rd well is our first colony that we have successfully completed ligated into psb1c3 Here you can see 4 lane in-between 25 and 55kDa Extra lane is our CAD1 enzyme denatured protein
To conclude, as you can see, the enzyme was not released in environment and we proved it in wells that we have loaded lysates next to precipitated ones.
Gel Results
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.
Design Notes
RBS and our protein coding region (ButCoaT) are digested from our whole construct with the enzymes SpeI, Sall, Xbal.
For ButCoaT ; E.Coli is synthesised suitable for Codon Bias. In the expression of pet28a (expression vector) , stop codon is removed in order to add HisTag.
Modelling
The figure above shows the increase in ButCoAT molecules over a time span of 10 seconds.
The figure above shows the molar increase in Butyrate concentration over a time span of 10 seconds.
Source
The ribosome binding site (B0034) that we’ve used is gotten from iGEM part library. Source of ButCoaT : Roseburia intestinalis L1-82
References
Hassig, C.A., Tong, J.K., Schreiber, S.L. (1997). Fiber-derived Butyrate and the Prevention of Colon Cancer
Roberto Berni Canani, Margherita Di Costanzo, Ludovica Leone, Monica Pedata, Rosaria Meli, Antonio Calignano. (2011). Potential beneficial affects of butyrate in intestinal and extraintestinal diseases
Duncan,S. H., Barcenilla, A., Stewart, C. S., Pryde, S. E., Flint, H. J. (2002). Acetate Utilization and Butyryl Coenzyme A (CoA):Acetate-CoA Transferase in ButyrateProducing Bacteria from the Human Large Intestine
Sequence : http://www.uniprot.org/uniprot/C7GB37?sort=score
Pathway : http://www.genome.jp/dbget-bin/www_bget?reaction+R00393+R01179+R01359+R07832
Picture : http://www.brenda-enzymes.org/structure.php?show=reaction&id=360609&type=S&displayType=marvin
RBS and our protein coding region (ButCoaT) are digested from our whole construct with the enzymes SpeI, Sall, Xbal.
For ButCoaT ;
E.Coli is synthesised suitable for Codon Bias. In the expression of pet28a (expression vector) , stop codon is removed in order to add HisTag.
Source
The ribosome binding site (B0034) that we’ve used is gotten from iGEM part library. Source of ButCoaT : Roseburia intestinalis L1-82
References
Hassig, C.A., Tong, J.K., Schreiber, S.L. (1997). Fiber-derived Butyrate and the Prevention of Colon Cancer
Roberto Berni Canani, Margherita Di Costanzo, Ludovica Leone, Monica Pedata, Rosaria Meli, Antonio Calignano. (2011). Potential beneficial affects of butyrate in intestinal and extraintestinal diseases
Duncan,S. H., Barcenilla, A., Stewart, C. S., Pryde, S. E., Flint, H. J. (2002). Acetate Utilization and Butyryl Coenzyme A (CoA):Acetate-CoA Transferase in ButyrateProducing Bacteria from the Human Large Intestine
Sequence : http://www.uniprot.org/uniprot/C7GB37?sort=score
Pathway : http://www.genome.jp/dbget-bin/www_bget?reaction+R00393+R01179+R01359+R07832
Picture : http://www.brenda-enzymes.org/structure.php?show=reaction&id=360609&type=S&displayType=marvin
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]
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