iGEM REC Chennai aims to tackle climate change with Project RECOVER, a hybrid carbon capture and biofuel production system. Our project involves the development of a system to capture flue gas and efficiently separate CO2 for utilization by engineered E. coli K12, producing Isobutanol in a reactor compatible with marine vessels. Achieving this milestone required several iterations in the engineering cycle consisting of the four stages below:
Design: Engineering principles are used to specify a biological system with an intended function, and models are used to help refine the initial design.
Build: The desired DNA sequence encoding the biological system or a part of the biological system is constructed and introduced to a chassis organism.
Test: The function of the engineered biological system is assayed.
Learn: The differences between the desired and observed function are analyzed, frequently with quantitative data, to develop an improved system.
In the design phase of our engineering cycle, we were committed to addressing the climate crisis by leveraging the power of genetically engineered E. coli K12. Our goal is to reduce carbon dioxide (CO2) emissions through the expression of specific enzymes, namely Phosphoribulokinase and Rubisco, within K12 that will actively utilize CO2. This critical step involves carefully planning and designing the genetic modifications that will enable E. coli K12 to efficiently Fix CO2, contributing to our mission of combating climate change.
To express PRK and RuBisCO in E. coli, we first codon-optimized the gene sequences of Synechococcus elongatus PRK and RuBisCO for RFC[10] to ensure efficient protein expression. Next, we performed restriction digestion and ligation, integrating the genes encoding PRK and RuBisCO into the pSB3K3 plasmid under the T7 promoter. Finally, the resulting plasmid was transformed into E. coli, completing this crucial step in our project.
We conducted the Xylose consumption growth curve for engineered E. coli that has RuBisCo(without chaperone - rbcX) and prk.
We initially observed the increase in growth curve made evidential that prk has been expressed and the xylose has been consumed and through the pentose phosphate pathway.By convention it generated Ribulose-5 phosphate. The expressed enzyme phosphoribulokinase which will convert Ribulose-5 phosphate into Ribulose-1,5 Bisphosphate which serves as a substrate for RuBisCO. We found growth arrest in E. coli over a period of time .
We had inferred from this experiment that the absence of functionality of RuBisCo lead to the accumulation of RuBP in the cell that lead to the cell death. Through the inputs from Dr. Natarajan we had been informed that the folding of proteins is important for the efficient functioning of an expressed enzyme.
In this design phase of our engineering cycle, our primary goal is to produce a fully functional RuBisCo enzyme along within K12 that will actively utilize CO2. This critical step was accomplished with the addition of a chaperone subunit in RuBisCo enzyme along with the large and small subunit
The codon optimized sequences of prk and the RuBisCo with chaperone was engineered into the pSB1C3 backbone.
We had successfully produced the RuBisCo enzyme and the functionality was proved by the Xylose utilization and efficient conversion of the xylose to pyruvate which was observed using a growth curve. The comparison between the engineered and control strain led us to a conclusion that addition of RuBisCo chaperone led to the functioning of the enzyme.
We initially observed the increase in growth curve made evidential that prk has been expressed and the xylose has been consumed and through the pentose phosphate pathway.By convention it generated Ribulose-5 phosphate. The expressed enzyme phosphoribulokinase which will convert Ribulose-5 phosphate into Ribulose-1,5 Bisphosphate which serves as a substrate for RuBisCO. We found growth arrest in E. coli over a period of time .
Lack of chaperone subunit is the reason for the problem in functionality of RuBisCo.
The addition of kivD and adhA parts in the plasmid for isobutanol production.
We engineered the dual plasmid prk +RuBisCo in psB1C3 backbone kivD+ adhA in psB3K3 backbone
We observed the production of isobutanol over a period of time which can be seen in the results
Efficient production of isobutanol was accomplished.