Team:SCAU-China/arcA-knock-out

Introduction

Escherichia coli possess the specific sensing/regulation systems for the rapid response to the availability of oxygen and the presence of other electron acceptors. The adaptive responses are coordinated by a group of global regulators, which includes one component Fnr (fumarate, nitrate reduction) protein, and the two-component Arc (anoxic redox control) system.

Principles

Arc system (contain ArcA as a effector and ArcB as a biosensor) is a feedback regulatory scheme under anaerobic condition, given the extensive repression of catabolic genes, including TCA cycle genes^[1]. Thus, both gene and enzyme level of the TCA cycle can be up regulated by arcB or arcA gene knockout under aerobic condition^[2] and anaerobic condition^[3]. It has been confirm that enhancement of TCA cycle in arcA knockout E.coli promote bioelectrocatalytic activity in microbial fuel cell^[4]. Therefore, we try to knock out the arcA gene of E.coli MG1655 in our project to explore the means to enhance electric generation in MFC.

λ-Red recombination system

Red recombination system is a well-known genetic manipulation tool in molecular biology, which contains three genes exo, bet and gam encoded by λ-phage genome with a function to activate homologous recombination between exogenous gene and bacterial genome in E.coli. This system is useful for chromosomal deletion in functional genomics.^[5]
pKD46 vector carries the red recombination system and temperature sensitive replicon. We knockouted our target gene arcA in E.coli MG1655 using pKD46 with following procedures (Fig. 1). The pKD46 vector was first transformed into target strain E.coli MG1655, followed by transform the PCR product containing a substitution fragment (Kanamycin resistance gene, NPTII) with 15-30 bp homologous arms from the upstream and downstream sequence of arcA gene in genome. With the modification of enzymes from red recombination system, genomic sequence arcA was finally substituted by NPTII sequence and lost during bacterial reproduction. arcA- knockout mutants can be screened in selection medium containing kanamycin.

Results

1.Knockout mutants were selected and further confirmed by colony PCR. We randomly chose 11 positive clones and used specific primers of arcA and NPTII gene respectively. The E.coli MG1655 containing vector pET-28a (+) (NPTII coding sequence) serves as a control.

2.NPTII sequence was than amplified using upstream and downstream primers of HA1 and HA2 (Fig.1) and sequenced to confirm its location. The sequencing result shows that it is identical to an expected sequence, indicating that the arcA gene was removed by NPTII from which it used to be. (Fig.3)
3. arcA mutant strain and wild type were cultured in anaerobic environment until reaching same optical density (OD600 nm= 2.0) and then transferred into MFC device (anaerobic anode chamber). Voltage and current data were collected every minutes and the measurement was conducted over a resistance of 1 kΩ. The electric charge of arcA mutant strain showed obviously higher electric charge than wild type one with a 317.15% increase.

References

[1] Federowicz S, Kim D, Ebrahim A, et al. Determining the Control Circuitry of Redox Metabolism at the Genome-Scale[J]. PLoS Genetics. 2014, 10(4): e1004264.
[2] Nizam S A, Zhu J, Ho P Y, et al. Effects of arcA and arcB genes knockout on the metabolism in Escherichia coli under aerobic condition[J]. Biochemical Engineering Journal. 2009, 44(2-3): 240-250.
[3] Nizam S A, Shimizu K. Effects of arcA and arcB genes knockout on the metabolism in Escherichia coli under anaerobic and microaerobic conditions[J]. Biochemical Engineering Journal. 2008, 42(3): 229-236.
[4] Liu J, Yong Y, Song H, et al. Activation Enhancement of Citric Acid Cycle to Promote Bioelectrocatalytic Activity of arcA Knockout Escherichia coli Toward High-Performance Microbial Fuel Cell[J]. ACS CATALYSIS. 2012, 2(8): 1749-1752.
[5] Datsenko, K. A. & Wanner, B. L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97, 6640-6645,(2000).