Team:Humboldt Berlin/Engineering

Engineering Success

Inducible Promoter System

In most synthetic biology projects, controllable and tunable transcription is crucial for the success. Often genetic circuits rely on a sharp and time resolved hierarchy of gene activation. For this, inducible promoters are an inventibly option to achieve the upper mentioned prerequisites. In our project, we also encountered the need to apply an inducible promoter for our expression system (link out to Pep8 expression). For this we first tried a construct containing an Arabinose inducible promoter ParaBAD fused to GFPmut2 (ParaBAD::GFPmut2) in the pGAP18 backbone. The araBAD promoter allows the L-arabinose induced expression of the fused construct.1 This way, gene expression can be controlled and adapted to the experimental requirements based on the applied L-arabinose concentration and glucose depletion.1 However, after successful cloning and transformation of the construct, no GFPmut2 expression was observed upon L-arabinose addition.

After extensive literature search, we concluded that potential uptake of Arabinose into minicells could be problematic due to the lack of importers. L-arabinose cannot penetrate the bacterial inner membrane.

Thus, L-arabinose intracellular uptake is transporter dependent by either a low affinity transporter araE (protein symporter) or high an affinity transporter araFGH (ABC-transporter).2 Furthermore, in contrast to the constitutively expressed and incessantly present low affinity transporter, the high affinity transporter is only formed upon L-arabinose sensing.2 However, minicells do not harbor a chromosome on which araFGH is encoded.

Also, and equally important, minicells possess limited transcriptional and translational capabilities due to their small size.3 We therefore consider the possibility that minicells are unable to effectively transcribe, translate and assemble the high affinity transporters in sufficient quantity. Finally, ATP is also limited in minicells which could further influence the effectiveness of ATP driven transporters in general.

Due to the presented reasons, we switched to a tetracycline-controlled transcriptional activation system (pTet-tetR) fused to our Salmonella codon harmonized GFP (BBa_K3861027; [BBa_K3861011 + BBa_K3861019]). The tetR system is well characterized and may be the most used gene expression controlling promoter system to date.4 There are two variants of this promoter-induction system, called Tet-On and Tet-Off.5,6 We use the Tet-On variant that selectively activates expression in the presence of tetracyclines. Important for our needs is the fact that tetracycline (or Anhydro-tetracycline) can passively penetrate bacterial membranes and no active transportes systems is crucial for this inducible expression system.

List of Sources

  1. Guzman, L. M., Belin, D., Carson, M. J. & Beckwith, J. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J. Bacteriol. 177, 4121–4130 (1995).
  2. Luo, Y., Zhang, T. & Wu, H. The transport and mediation mechanisms of the common sugars in Escherichia coli. Biotechnol. Adv. 32, 905–919 (2014).
  3. Farley, M. M., Hu, B., Margolin, W. & Liu, J. Minicells, Back in Fashion. J. Bacteriol. 198, 1186–1195 (2016).
  4. Gossen, M. & Bujard, H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc. Natl. Acad. Sci. U. S. A. 89, 5547–5551 (1992).
  5. Baron, U. & Bujard, H. Tet repressor-based system for regulated gene expression in eukaryotic cells: principles and advances. Methods Enzymol. 327, 401–421 (2000).
  6. Berens, C. & Hillen, W. Gene regulation by tetracyclines. Constraints of resistance regulation in bacteria shape TetR for application in eukaryotes. Eur. J. Biochem. 270, 3109–3121 (2003).