Team:GreatBay SZ/Design and result/e-PNs Plasmid Construction
The Key e-PNs Plasmid Construction
Why do we need to express the e-PNs in the E.coli?
The original bacteria that expressed e-PNs is an Archaea — Geobacter sulfurreducens, which needed to be routinely cultured at 25 °C under strict anaerobic conditions.[1]
Technical constraints limited mass cultivation and genetic manipulation of G. Sulfurreducens.
What is more, e-PNs expressed in E. coli had the same diameter (3 nm) and conductance as e-PNs expressed in G. Sulfurreducens. These results, plus, E.coli as a commercialized bacteria, associated with its robustness for mass cultivation and the comprehensive E.coli toolbox for genetic manipulation, made it convenient for our work on protein optimization, such as constructing RBS libraries and engineering e-PNs to boost the efficiency of generating electricity. Therefore, we decided to express e-PNs in E.coli. [2]
Overview
The target genes we intended to assemble in the plasmid (pET24b[2]) included a tac promoter, a pilA gene from Geobacter sulfurreducens which expressed the monomer of the pili fused with the PpdD signal sequence and a group of genes responsible for the secretion of type IV pili[2].
We separated the genes added into six parts.
Cloning Part I&II
Both part I and II were constructed without template by using PCR fusion with specific primers. Due to the restriction on length of primers related to the speed of synthesis, we designed two pairs of primers for part I and three for part II respectively, ending up successfully obtaining the two parts.
Cloning Part III to VI and the Vector
Part III to V and VI were acquired using PCR from E.coli O157:H7 and MG1655, respectively. The linearized pET24b vector was obtained in the same way as well.
Assembling the Fragments
Later, in order to shun the inefficiency of Gibson Assembly when connecting 6 separate parts and a vector, we first combined part I, II, and III into a larger fragment, and part V, VI into another using PCR fusion, with the result that we only needed to connect three fragments(part I-II-III, part IV and part V-VI)and the vector together.
We then assembled part I-II-III, part V-VI and the vector with Gibson, since the length of part IV(3330 bp) was too long for efficient assembly. The insertion of part IV was saved as the last step, in which we linearized the plasmid we obtained from the last step and connected it with part IV using Gibson again.Our Results
The sequence of each part on the plasmid was examined and the result turned out that there was a mutation on the hofB site. Thus, we designed two pairs of primers: one for modifying the mutation site; The other pair, designed at the Kana resistance gene site, was used to improve the efficiency of screening and avoid cloning too long fragments. Eventually, after agarose gel electrophoresis (AGE), we got a perfect version of e-PN plasmid with the essential genes for the production of type IV pili, which was ready to be transferred into the target E.coli △fimA for the production of protein nanowires.
Figure 2: From left to right: Vector1A Vector1B Vector1C Vector1D Vector2A Vector2B Vector2C Vector2D Vector3A Vector3B Vector3C Vector3D Vector4A Vector4B Vector4C Vector4D
AGE Map, which demonstrated our successful results of hofb site modifying.
Reference
[1]Liu X, Gao H, Ward J E, et al. Power generation from ambient humidity using protein nanowires[J]. Nature, 2020, 578(7796): 550-554.
[2] Ueki T, Walker D J, Woodard T L, et al. An Escherichia coliChassis for Production of Electrically Conductive Protein Nanowires.[J]. ACSSynthetic Biology, 2020, 9(3): 647-654.