Measurement

Expression Burden

In synthetic biology, a common issue in integrating exogenous sequences into host cells is that heterologous gene expression can cause significant metabolic burden. Typically, this gene expression burden is implied by a declining growth rate of bacteria, which is unfavorable for fermentation industry because it lowers yields and profits. According to Ceroni et al. (2015), a fluorescence-based method allows to measure gene expression capacity of cells, by inserting a “capacity monitor”.

Capacity Monitor

The “Capacity Monitor” consists of a superfold GFP with a strong constitutive promoter, which is constantly expressed in the bacterial genome. Thus, any alternation in fluorescence intensity can reflect in overall resource availability in the cell. Particularly, with expression of synthesized genes, the expression of GFP will decrease, thus reporting a reallocation of cellular resources.

VioABDE

After applying capacity monitor, we compared the fluorescence of E. coli with an empty vector and E. coli with VioABDE inserted and expressed. Both strains were incubated for 24 hours with fluorescence and OD constantly monitored. However, the OD of bacteria with VioABDE increased faster than the bacteria without Vio expression, which contradicts our initial expectation.

We co transformed the TlpA39-VioABDE into the capcatiy monitor component cell which was made by beyotime component cell kit.

Then the Capacity Monitor and Capacity Monitor-Vio were incubicated in microshaker in 39 degree Celsius, then they were added to the 96 well plates. "

Alternative Device

Since we target to modify bacteria used in fermentation industry, which primarily are used for material synthesis and production, our sequence inserts should occupy as limited resources in bacteria as possible to avoid significant expression burden. In order to reduce this expression stress, we designed another device for fermentation which used a LuxR repressive promoter (Peking-S, 2011) and the “Cold Box” in 5’UTR region of CspA (Ionis Paris, 2017). With only one transcriptional regulator, less energy will be consumed, which is beneficial for fermentation industry.

References

Ceroni, F., Algar, R., Stan, G. B., & Ellis, T. (2015). Quantifying cellular capacity identifies gene expression designs with reduced burden. Nature methods, 12(5), 415.

Ionis Paris. (2017). Biological thermo-responsive solution, that reduces the plant’s thermal stresses. Available from: https://2017.igem.org/Team:IONIS-PARIS.

Peking-S iGEM. (2011). A Chemical Wire Toolbox for Synthetic Microbial Consortia. Avaible from: https://2011.igem.org/Team:Peking_S.

Team:RDFZ-China/Measurement