Novel Temperature-Dependent Readout
We were interested in the construct originally created by Heidelberg where they had stuck together a valine and indigoidine module in order to produce a Val-Ind dipeptide. The dipeptide was only confirmed by TLC spot and we wanted to investigate whether we could identify it via MS. Our goal was to verify dipeptide production and use it as a starting platform to rapidly investigate the modification of NRPS module substrate specificity and substrate modification.
When we first received the BioBrick we had plated it and left the agar stab at room temperature over night. Upon returning the next day we found that the agar stab had turned a blue colour canonical to the production of indigoidine.
We had attempted to transform the plasmid into BAP1 E. coli which contains a gene for SFP, a promiscuous PPTase from B. subtilis. However after many failed attempts and running several positive and negative controls we were still unsuccessful at expressing the Val-Ind BB in BAP1 E. coli. We hypothesized that the T7 Promoter which controlled PPTase production was leaky. This was in part due to the leaky nature of the Lac promoter by which T7 Polymerase was controlled. Efficient activation of IndC may have lead to abundant IndC production which was arresting cell growth.
It was noted in literature that E. coli did not possess the PPTase required for IndC activation. It had always been supplemented with a sfp gene. In order to verify that it was indeed indigoidine production negatively impacting our transformations we had transformed BL21 E. coli with the Val-Ind biobrick.
The E. coli grew well at 37°C over night and were taken out of the incubator after 16 hours and left at room temperature before being placed at 4°C. Within 20 minutes of being at room temperature (22°C) they had begun to turn a blue hue (below left). When placed in 4°C however they had rapidly tuned into the brilliant blue colour (below right).
It has been postulated that the EntB PPTase gene found in E. coli is able to activate the indigoidine module. What is more interesting is that the production of indigoidine is temperature dependent. This would allow one to use indigoidine producing biobricks to visualize a decrease in temperature.
References
1. Hahn, M., & Stachelhaus, T. (2004). Selective interaction between nonribosomal peptide synthetases is facilitated by short communication-mediating domains.
Proceedings of the National Academy of Sciences of the United States of America, 101(44), 15585-15590.
2. Takahashi, H., Kumagai, T., Kitani, K., Mori, M., Matoba, Y., & Sugiyama, M. (2007). Cloning and characterization of a Streptomyces single module type non-ribosomal peptide synthetase catalyzing a blue pigment synthesis.
Journal of Biological Chemistry, 282(12), 9073-9081.
3. Marahiel, M. A., & Essen, L. O. (2009). Nonribosomal peptide synthetases: mechanistic and structural aspects of essential domains.
Methods in enzymology, 458, 337-351.
