The Aim
We want to create a reliable quantification method for SCFAs, which is not only cheaper but also more accessible than the conventional methods. For this we created several E. coli strains that express fluorescent proteins when they detect short chain fatty acids, we came up with three different constructs for the three most common SCFAs in the human body (acetate, propionate and butyrate).
Our Constructs
The constructs are based on the vector pBluescript KS+ and consist of a promotor that activates upon binding directly to the SCFA or to metabolic derivative of one. This promoter then induces the transcription of a fluorescent protein whose fluorescence we can then detect.
In total we created 9 such constructs:
| # | Name | Promotor | Fluorescent Protein | Part Number |
|---|---|---|---|---|
| 1 | Acetate-GFP | GlnAp2 | GFP | BBa_K4930014 |
| 2 | Acetate-BFP | GlnAp2 | BFP | BBa_K4930015 |
| 3 | Acetate-RFP | GlnAp2 | RFP | BBa_K4930016 |
| 4 | Propionate-GFP | prpB | GFP | BBa_K4930017 |
| 5 | Propionate-BFP | prpB | BFP | BBa_K4930018 |
| 6 | Propionate-RFP | prpB | RFP | BBa_K4930019 |
| 7 | Butyrate-GFP | pchA | GFP | BBa_K4930020 |
| 8 | Butyrate-BFP | pchA | BFP | BBa_K4930021 |
| 9 | Butyrate-RFP | pchA | RFP | BBa_K4930022 |
Acetate
For the acetate-inducible system the glnAP2 promoter is used, which is normally induced by the NRII kinase under nitrogen deficiency. By inactivating the NRII kinase, the glnAP2 promoter can be induced by acetyl phosphate, which is produced from acetate by the acetate kinase. Furthermore, by inactivating the phosphotransacetylase in E. coli, the influence of the intracellular acetate metabolism on the promoter can be reduced and the promoter can be made dependent on exogenous acetate. Cloning a reporter gene under the control of the glnAP2 promoter makes the quantification of acetate possible, whereby the the emitted fluorescence represents the concentration of acetate in a given sample.1
Propionate
In order to create a propionate-inducible expression system, a series of expression vectors (pPro)
have already been constructed for the regulated expression of genes in E. coli. The pPro vectors
contain the prpBCDE promoter (P(prpB)), which is responsible for the expression of the propionate
catabolism genes (prpBCDE). Additionally, they encode prpR, which acts as a positive regulator of
this promoter. In E. coli, propionate is converted to 2-methyl citrate (2-MC) in two consecutive
steps. In the presence of 2-MC, PrpR initiates transcription of prpBCDE by binding to an
enhancer-like element located at a distance 5’ to the prpBCDE promoter and contacting the RNA
polymerase by DNA loop formation.
This already established system is used to create a reporter construct enabling the
quantification of propionate in a concentration-dependent manner by cloning a fluorescence
reporter protein downstream of the prpB promoter. Thus, the emitted fluorescence represents
the concentration of propionate in a given sample.2
Butyrate
The PpchA promoter is used for the butyrate-induced construct. This promoter regulates the transcription of the pchA regulator, which is responsible for the activation of the transcription of the locus of enterocyte effacement 1 (LEE1) operon in enterohemorrhagic Escherichia coli (EHEC). Butyrate forms a complex with the native E. coli leucine-responsive regulatory protein (Lrp) and can thus bind to the promoter of the pchA gene. By cloning a fluorescence reporter gene downstream of the pchA promoter, we want to create a butyrate-inducible reporter system.3
References
[1] Farmer, W. R. & Liao, J. C. Acetate-inducible protein overexpression from the glnAp2 promoter of Escherichia coli. Biotechnol Bioeng 75, 504–509 (2001)
[2] Lee, S. K. & Keasling, J. D. A propionate-inducible expression system for enteric bacteria. Appl. Environ Microbiol 71, 6856–6862 (2005).
[3] Takao, M., Yen, H. & Tobe, T. LeuO enhances butyrate-induced virulence expression through a positive regulatory loop in enterohaemorrhagic Escherichia coli. Mol Microbiol 93, 1302–1313 (2014).