Team:ICT-Mumbai/Results
The big picture
As described in Description, we wish to construct Escherichia coli cells that can assimilate ammonia and convert it into an innocuous substance. As the first step, we decided to overexpress the native enzyme glutamine synthetase, which condenses ammonia with glutamate to form glutamine.
Cloning glnA in pET43.1b
The glnA gene codes for the enzyme glutamine synthetase. We amplified glnA from the E. coli MG1655 genome and cloned it in plasmid pET43.1b between the NdeI and HindIII sites to form plasmid pET43-glnA.
Co-expression of indC
The gene indC codes for blue pigment synthase (Bps), which converts glutamine to the blue-colored compound indigoidine. Plasmid pRB5, which was obtained as a kind gift from Team Heidelberg, carries the indC gene under control of a lacI-regulated promoter.
We decided to co-transform plasmids pET43-glnA and pRB5 in E. coli BL21(DE3). However, we realized that both these plasmids have the same origin of replication (from pMB1), and would therefore not be maintained in a single cell. To circumvent this problem, we decided to clone glnA in a plasmid that has a different origin of replication.
Cloning glnA in pSEVA234
Plasmid pSEVA234 has an oriT origin of replication, and therefore will be able to co-exist with plasmids that have a pMB1 origin of replication. To clone glnA in pSEVA234, pET43-glnA was digested with Psp5II and XhoI to release the lacI-T7 promoter-glnA fragment. This was blunted and cloned in SmaI-digested pSEVA234 to form plasmid pSEVA234-glnA.
Demonstrating proof-of-concept
To demonstrate proof-of-concept, we have transformed E. coli BL21(DE3) with
- plasmid pRB5
- plasmids pRB5 and pSEVA234-glnA
E. coli BL21(DE3) transformed with both plasmids is expected to produce indigoidine at a faster rate, as compared to E. coli BL21(DE3) transformed with pRB5 alone.
Biobricking ychH promoter
As described in Parts, we wish to express enzymes for ammonia synthesis using a constitutive promoter that is active under nutrient starvation conditions. For this purpose, we chose the promoter of the ychH gene. We amplified a fragment containing the ychH promoter from the E. coli MG1655 genome using the following primers:
- Fwd_BB_ychH_prom: 5’-GTTTCTTCGAATTCGCGGCCGCTTCTAGAGgtttttttgtcctgagtgtgtacataac
- Rev_BB_ychH_prom: 5’-GAAGAAACCTGCAGCGGCCGCTACTAGTAtcacctccggaactttctg
Primers Fwd_BB_ychH_prom and Rev_BB_ychH_prom contain the prefix and suffix, respectively, of a RFC10-compatible BioBrick. The BioBricked ychH promoter, along with the native RBS, was cloned in EcoRI- and PstI-digested pSB1C3 and submitted to the iGEM Parts Registry as Part BBa_K2479000.
Biobricking glnA gene
The glnA gene is cut by EcoRI at two places. To BioBrick this gene, these sites have to be removed. We got glnA gene with modified EcoRI sites synthesized as two 1 kb gBlocks from IDT; the first gBlock carried a 3' sequence homologous to the 5' sequence of the second primer. The two gBlocks were joined together by carrying out SOE PCR using Phusion DNA polymerase. However, multiple attempts to join the two gBlocks were unsuccessful.
Getting E. coli to work
The enzyme glutamine synthetase requires ATP as a cofactor, and releases protons on catalyzing the formation of glutamine from ammonium and glutamate. Hence, ATP will have to be continuously supplied to keep on assimilating ammonium. This can be achieved by pumping out the protons formed using proteorhodopsin, which is a light-powered proton pump. The resulting proton gradient can then drive native ATP synthase to form ATP, required for glutamine synthetase activity.
Proteorhodopsin requires retinal for activity. Retinal can be synthesized by E. coli when transformed with part BBa_K1604022. This part contains five genes under control of an arabinose-inducible promoter that code for enzymes that catalyze the formation of retinal starting from FPP farnesyl diphosphate.
To demonstrate that our idea works, E. coli will be transformed with two plasmids — BBa_K1604022, and a plasmid that contains genes coding for glutamine synthetase, blue protein synthase, and proteorhodopsin under control of an inducible promoter. As activity of proteorhodopsin is dependent on light, the cells will be exposed to a source of visible light.
Final construct...
We look forward to expressing all genes required to assimilate ammonium into indigoidine from the ychH promoter as a single operon. To prevent metabolic burden on the cell, instead of transforming E. coli with multiple plasmids, we will integrate all the genes in the genome, thereby also eliminating use of antibiotic-resistance genes to maintain plasmids within the host cells.
