The Sustainability Suite
iGEM Hamburg proudly presents: The Sustainability Suite. Bacteria have a straight forward evolutionary strategy: Grow faster than you die. This benefits molecular biology, as it makes working with bacteria easy. However, this biology becomes a pitfall for elaborate systems designed for a specific function. Especially systems desired to function well for a long time without maintenance, like many synthetic biology applications for use in the field, are practically impossible to implement outside of the laboratory. With the Sustainability Suite we provide a toolset to monitor and respond to nutrient availability with both positive and negative transcriptional signals, allowing users to control metabolic load under varying circumstances. With the included cell growth regulators, bacteria cultures can be kept stable for extended periods of time without maintenance. We even provide a complete synthetic operon limiting bacterial growth, allowing your bacteria to fully concentrate on what you want it to do.
Monitoring and Responding to varying nutrient availabilities
To control the metabolic load of bacteria under differing conditions, two different goals are very important. On the one hand, a glucose-dependant regulation is wise, if genes are wanted to be active at high glucose concentrations and inactive without glucose. This is very useful, if cells shall be sustained for a long period of time. Thus, great metabolic burdens can be prevented, e.g. by shutting down energy-consuming protein production when no glucose is present.
On the other hand, regulating gene expression by a contrary approach is also a possible approach.
Although many genes in E. coli are regulated in a glucose-dependant manner, it was quite hard to find candidates for such a promotor, but finally, we decided to use MlcRE. After characterizing this part, it was clear that this promotor does not have the desired properties, as it usually shows an increased activity at low glucose-levels. Nevertheless, this part can be easily used in case of genes that should be active at low or even absent glucose levels.
Searching for a promotor with the same characteristics, we planned to use a NOT-gate based approach including the RnaG120 operator for generating a glucose-dependant promotor. This part is comprised of several subparts in 5’ to 3’ order: BBa_B0015 terminates transcription of RnaG120. The constitutive promoter MlcRE provides constant gene of interest (GOI) mRNA transcription. icsA 5’ UTR, which is complementary to RnaG120, acts as 5’ UTR for any GOI downstream of RnaG120-based NOT-gate of MlcRE, sent in as BBa_K2588002. MlcRE is cloned in opposite reading direction, downstream of icsA 5’ UTR, activating transcription of RnaG120. Downstream of BBa_K2588002, any gene of interest could be placed.
Regulation of growth by a novel growth inhibition module
In addition, we designed a growth inhibition module consisting of several genes acting on different aspects of cell division. With this, bacteria cultures can be kept stable for extended periods of time without maintenance. We proved that our module BBa_K2588021 is well performing; E. coli cell division rate was reduced to a third. At this point, the composite part consists of mraz, cbtA and cspD. cptA is a protein, which inhibits cell division by preventing formation of the ftsZ ring, whereas mraZ constrains synthesis of peptidoglycan. Since another approach is the inhibition of DNA synthesis, we chose cspD, a protein which interferes with elongation of DNA during DNA synthesis.