RECOVER" from iGEM REC-CHENNAI represents a remarkable advancement in synthetic biology. Building upon our previous success in project "curlim", we team have achieved significant milestones in addressing environmental and energy challenges. This year in our project "RECOVER" we meticulously crafted four major functional parts, following RFC 10 standards, revolutionizing the field of genetic engineering. These parts are pivotal in solving our problem statement "CO 2 Emission" through two distinct modules. For the first module, major composite part specifically BBa_K4689369, was constructed to address carbon dioxide (CO2) fixation and utilization. By ingeniously engineering CO 2-fixing enzymes like RUBISCO and PRK, CO2 is converted into pyruvate, a crucial substrate for the subsequent module. This pioneering approach holds the promise of atmospheric CO2 level reduction and climate change mitigation. In the second module, a composite part, BBa_K468936, with a focus on sustainable biofuel production, was engineered. Leveraging the keto acid pathway with a minimum bioburden of our organism and meticulously regulating enzyme expression, the transformation of pyruvate into isobutanol, a valuable biofuel, was successfully achieved.

CO2 FIXATION PHASE

Outline: In our CO2 fixation module, we introduced two pivotal non-native enzymes, PRK and Rubisco, into the E. coli organism. PRK facilitates the conversion of ribulose-5-phosphate (Ru5P) into ribulose-1,5-bisphosphate (RuBP), while Rubisco plays a crucial role in carbon dioxide fixation and the subsequent conversion of RuBP into 3-phosphoglycerate (3PG). We meticulously engineered the composite genetic part, BBa_K4689369, to construct our CO 2 fixation system. When this part was integrated into the pSB3K3 backbone, both PRK and Rubisco were successfully expressed, as demonstrated by the methyl red test, which revealed an increased production of pyruvate compared to the natural bacterial phase. This excess pyruvate serves as a crucial substrate for our subsequent module.


Name Type Description Length(bp) Notes Organism
BBa_K4689369 pT7-rbs-rbcL-rbs-rbcS-rbs-rbcX-rbs-prk-double terminator Composite part 3386 prk and RuBisCO under T7 promoter E.coli-K12

Isobutanol Production phase

In our isobutanol module, we undertook the genetic engineering of two significant enzymes, KivD and AdhA, with distinct origins in our organism. KivD, originating from a non-native source, emerged as the linchpin in our process, as it played a pivotal role in converting 2-Ketoisovalerate into isobutyraldehyde. This biochemical pathway commences with pyruvate, a central player in cellular energy metabolism, highlighting the integration of our engineered system into the organism's fundamental processes. Meanwhile, adhA, an intrinsic enzyme inherent to E. coli, took center stage in the subsequent step, catalyzing the transformation of isobutyraldehyde into isobutanol. To streamline our system, we designed a composite genetic part, BBa_K4689361, which encompassed both KivD and adhA. This composite component was seamlessly integrated into the PSB1C3 vector, enabling us to monitor the expression of our engineered enzymes. Our assessment involved the quantitative analysis of isobutanol production using high-performance liquid chromatography (HPLC), serving as a reliable indicator of the effectiveness of our genetic modifications and confirming the functionality of the incorporated genes.


Name Type Description Length(bp) Notes Organism
BBa_K4689361 pT7-RBS-kivD-RBS-adhA-Double terminator Composite part 2849 KivD and adhA under T7 promoter E.coli-K12