Part:BBa_K4930019

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 (Lee & Keasling, 2005).

This already established system is used to create a reporter construct enabling the quantification of propionate in a concentration-dependent manner by cloning RFP (BBa_K4930007) downstream of the prpB promoter (BBa_K4930004). In addition, prpR (BBa_K4930008) is introduced into the plasmid backbone, in order to fulfill it's function as the positive regulator of the prpB promoter. Thus, the emitted fluorescence represents the concentration of propionate in a given sample.

The Glowcoli Tag (BBa_K4930010) is a made-up sequence acting as a spacer between the promoter and the coding sequence. Right after the Glowcoli Tag we integrated the Shine-Dalgarno-Sequence (BBa_K4930009), which plays a crucial role in bacterial translation (Malys, 2012).

Validation

We followed the Gibson protocol as before and used the same plasmid. Prior to streaking the transformed bacteria on the plates, we spread X-Gal (5-Bromo-4-chloro-3-indolyl-β-D-galactoside) on the LB agar plates. This allowed us to perform a blue-white selection. For blue-white selection, a plasmid can be used that contains the gene for β-galactosidase (lacZ gene) at the position where the transgene is inserted into the plasmid (Multiple Cloning Site). Inserting an insert into the Multiple Cloning Site inactivates the galactosidase. As a result, after transformation, the transgenic organisms, in contrast to the non-transgenic organisms, do not contain a functional galactosidase, causing their colonies to remain white, allowing to be isolated based on their lack of color.

As seen in the figure above, both white and blue colonies grew. So we used a pipette tip to pick the white colonies and subsequently conducted another Colony-PCR. As the figure demonstrates, bands in the Propionate constructs are visible and correspond to the expected band lenghts.'Pro' stands for the primers that only amplify the promoter, while 'Rep' represents the primers that amplify the respective reporter genes. The insert containing the Propionate promoter includes an additional spacer to extend the short promoter with 62 base pairs to 252 base pairs. In the GFP and BFP constructs, a band at approximately 250bp can be detected, which supports the amplification of the correct band. Additionally, in the wells containing the primers for the reporter genes, bands with 750 base pairs can be observed. Since the reporter genes, GFP, BFP, and TaqRFP, all range between 740 and 750bp, this band also indicates a successful amplification of the desired insert.

The figure below displays the Colony-PCR using primers for the regulatory sequence prpR, which is 1616bp long. A prominent band is visible in the RFP construct, which does not exist in the other constructs (exceeding 5000bp). As the entire construct, including the promoter and regulatory sequence, is 5702bp in length, the amplification of the entire construct cannot be ruled out. Overall, it can be concluded that the Colony-PCR for Propionate suggests that the Gibson Assembly worked. Due to a lack of time, planned experiments with the successful Gibson were not conducted in a timely manner but will be continued in the future.

Lee, S. K. & Keasling, J. D. A propionate-inducible expression system for enteric bacteria. Appl Environ Microbiol 71, 6856–6862 (2005).

Malys, N. Shine-Dalgarno sequence of bacteriophage T4: GAGG prevails in early genes. Mol Biol Rep 39, 33–39 (2012)

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 2520
  Illegal SapI.rc site found at 1902