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LINKS_China established a collection for electricity conductive pili (e-pili) production which are pilis that can produce electricity within humid environments. We first synthesized and constructed three major pilin genes of types of e-pili: GsPilA, PaPilA and GmPilA. Second, we amplified the gene cluster of pili generator system which is for the expression of e-pili from E.coli DH5a, and conducted point mutation to derive them into genes in EHEC for increasing the e-pili production. Here we offered two successful generator combinations from EHEC and DH5a for e-pili production. Next, we decoupled the 12 genes of our generator and reconstructed each gene after codon optimizations. Finally, we produced the e-pilis and verified their functions of producing stable and sustainable voltage and current. We hope this collection may provide future teams in the iGEM community with the tools for more e-pili discovery.
Our part collection can instruct other teams to designed new rechargeable pilus and substitution of different major pilin.
To get a complete pili production system, we first ordered oligo DNAs from pilA gene and assembled them by SOE PCR to acquired its completely same amino acid sequence as expressed by EHEC genes. Then we constructed the generator of the pili and obtain a complete circuit of Gs pilA. We also discovered 2 new pilA, Pa pilA and Gm pilA and we constructed them as the same method as Gs pilA.
We amplified each gene cluster of pili generator (hofBC, hofMNOPQ, and ppdAB-ygdB-ppdC-gspO) from E.coli DH5a. Then, due to the long length of each gene cluster, we initially assembled all segments we obtained separately on pSB1CJ vector by Gibson Assembly to form three sub-plasmid: 1)Gs pilA and hofBC, 2)hofMNOPQ, and 3)ppdAB-ygdB-ppdC-gspO. Additionally, we constructed another sub-plasmid with pET28a vector for the backbone of our major plasmid. Finally, four sub-plasmids were assembled through Golden Gate Assembly to form the final major plasmid of pilA with pili generator.
Since using the pili generator from the strain Enterohaemorrhagic Escherichia Coli (EHEC) will give a better production of T4P, we conducted point mutation on three the sub-plasmids we constructed before for further construction, deriving the original gene sequence into that of EDL933, obtaining three new sub-plasmids. We then conducted the same assembling method to assemble three new sub-plasmids on pET28a vector into a new major plasmid possesses Gs pilA with EHEC pili generator.
For pili expression, we chose E.coli BL21 because it is the best fit to our project as it has the highest pili yield among them and is easy to obtain. On the cultivation part, we cultured the bacteria in solid M9 medium. Additionally, we provide the bacteria with glycerol as the carbon source because it was confirmed to help improve the conductivity of the pili produced.
We transformed the plasmid with pilA and generator into E.coli BL21. The bacteria was first cultivated on a LB medium plate with kanamycin at 30°C for 24h. Then, we scraped the E.coli off the LB plates with 6ml liquid M9 solution and then coated the collected bacteria solution on 20 M9 medium plates with IPTG and kanamycin. Finally, the 20 coated M9 plates were cultured at 30°C for 48h. We harvested the bacteria from M9 plates after 48h cultivation by scrapping them off again with liquid M9 solution and collect the bacteria solution. By conducting the extraction and purification on the samples, we managed to get an amount pili solution dissolving in 150mM ethanol-amine after filtration with a 100kDa membrane in nitrogen gas.
We then took a SEM photography of the pili to confirm their structure.
We also conducted experiments to measure the voltages of each batteries. First we manufactured six standard electrodes by using three pili, two pieces each, all with triple layer of pili covered. Then we measured the voltage and compared the result which shows that Gm pili has the highest value of measured voltage where Gs pili has the least.
In order to compare the pili yield, we established a new measurement for a quicker, clearer, and more accurate determination of pili production. We use his-tags antibodies to attach on the his-tags on the pili, and then the secondary antibodies will attached to the his-tags antibodies for coloration directly on the outer membrane of the bacteria. We analyse the datas and included the effects of two variables on pili production: cultivation time and mobile oxygen presence. The result shows a ranking from Gs pili to Pa pili to Gm pili.
We carried out decoupling of all the genes into single parts. We mutated the sequence and codon optimization of the repeated sequence from two genes to prevent recombination. In the future we are going to link all single genes to the same promotor and reconstructed all genes into a complete circuit to seek for any difference comparing to the initial combined genes.