Wenxuan Chen, Lili Yin, Chao Shen, Yufei Zhang, Lujie Liang, Ziyan Huang, Taidou Hu,

Qian Nie, Faming Chen, Yonghao Shi, Chao Zheng, Ruite Chen, Wenyi Zhang, Wencong Zhu

College of Life Sciences, South China Agricultural University 510642, China

Our project aims to develop new solutions

for rapid depletion of fossil energy resources

and increasing shortage of fresh water. We

genetically modified E. coli with two

strategies: (1) knocking out a tricarboxylic

acid cycle (TCA) suppressor arcA to boost up

TCA cycle under anaerobic condition; (2)

over-expressing NAD synthetase encoding

gene nadE to enhance the NAD+ production.

Both increase the yield of electron carrier

NADH and result in higher electrogenic

capacity in microbial fuel cell (MFC).

Moreover, the modified MFC was coupled

with seawater desalination to acquire fresh

water in a microbial desalination cell (MDC).

Together, our project enables more fresh

water and electricity in M D C or MFC,

respectively .

◆ NAD+(H) pool plays a central role in intracellular

redox state of electricity-producing bacteria. nadE

encoding NAD synthetase limits the NAD production

leading to few intracellular electron carrier NADH in cells.

NAD synthetasee-

e-

e-

◆ Over-expression of nadE to increase

NAD+(H) level for more intracellular electrons in

MFC or MDC.

Overlap Sequence on BioBrick suffix

METHODOLOGY

nadE

F-K608002-nadE

R-suffix-nadE

Genomic DNA of E.coli MG1655

5'

3'

3'

5'

nadE

BBa_K608002

BioBrick Prefix BioBrick Suffix

pSB1C3

nadE

nadE

3'

3'

5'

5'

1stΩ-PCR Overlap Sequence on K608002

PCR

2ndΩ-PCR

Mega-primers

BBa_K608002BioBrick Prefix BioBrick Suffix

pSB1C3

nadE

Insertion site

BioBrick Construction

Procedures of arcA knock-out with

Red Recombination System.

New BioBricks for overexpression (e.g. nadE)

were constructed via Ω-PCR

◆With the red recombination system,

genomic arcA was replaced by NPTII.

The arcA knock-out mutants were

obtained from kanamycin selection

medium.

◆ Existing BioBricks with constitutive

promoters were fused with nadE via

Ω-PCR to generate constructs for

na dE o ve r e xp r ess i o n driven by

different promoters.

NAD+

NADH

16S

nadE

Fig. 2 (A) mRNA level of nadE in semi-quantitative RT-PCR assay. Strains with BBa_K1373001

and BBa_K1373002 shows higher level than wild type. (B) Protein level of nadE in SDS-PAGE.

Extra bands present in the strains with BBa_K1373001 and BBa_K1373002 , the protein levels are

consistent with those in mRNA level.

nadE

29 kDa

nadEnadE

Overexpression of nadE is confirmed in mRNA and protein level

Fig. 3 (A) Voltage data of wild type (MG1655) and nadE overexpression transgenic stains with vector

BBa_K1373001. nadE over-expression results in 1 fold higher maximal voltage output than the wild type

one with a peak value 172.09 mV. (B) Electric charge yield in wild type, nadE-overexpressed

BBa_K1373002 and BBa_K1373001 in 700 minutes. The nadE-overexpressed strain driven by strong

promoter (BBa_K1373001) produce approximately 738.60% electric charge than the wild type, while the

one with weaker promoter (BBa_K1373002) increase 433.30% electrical energy than the wild type.

Expert consultations:

◇ Project feasibility evaluation.

◇ Biosafety assessments.

◇ MFC device assembly.

Questionnaire:

◇ Public awareness of synthetic biology.

◇ General attitude to biological energy.

◇ Opinions and suggestions to our project.

◇ Safety concerns of biological industry.

Scientific Exhibition:

◇ Popularization of synthetic biology by promoting

its applications.

◇ Project promotion.

High School Teach-ins:

◇ Lectures on synthetic biology.

◇ Discussion on biosafety issues with younger minds.

iGEMers Meet up:

◇ Experience exchanges.

◇ Laboratory visiting.

◇ Friendship and cooperation.

INTRODUCTION PRINCIPLES & DEVICES

RESULTS

Fig. 1 (A) Colony PCR result. Knockout mutants were confirmed by colony PCR and sequencing,

which

proved arcA was replaced by NPT II gene from its original location. (B) Electric charge yield in wild

type and △arcA in 700 minutes. arcA mutant strain showed obviously higher electric charge than

wild type one with a 317.15% increase.

◆ Eight BioBricks were successfully constructed and submitted to the

iGEM data base.

◆ Over-expression of nadE and knock-out of TCA suppressor arcA

achieved better performance in MFC. Thus genetic modification of

microbes is a promising way to improve intracellular electron yield.

◆ Our MFC-based MDC system is able to turn seawater to fresh water.

◆ Our project raise public awareness on new energy resources.

②

1.Chen L, Wang F, Wang X, Liu YG. Robust one-tube Ω-PCR strategy accelerates precise sequence modification of

plasmids for functional genomics. Plant Cell Physiology. 2013, 54: 634-642

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.Yong, X.-Y. et al. Enhancement of bioelectricity generation by cofactor manipulation in microbial fuel cell. Biosensors and

Bioelectronics. 2014, 56: 19-25.

4.Hatzell, M. C. and B. E. Logan. Evaluation of flow fields on bubble removal and system performance in an ammonium

bicarbonate reverse electrodialysis stack. Journal of Membrane Science. 2013, 446(0): 449-455.

5.Kim, Y. and B. E. Logan. Series Assembly of Microbial Desalination Cells Containing Stacked Electrodialysis Cells for

Partial or Complete Seawater Desalination. Environmental Science & Technology. 2011, 45(13): 5840-5845.

6.Kim, Y. and B. E. Logan. Microbial desalination cells for energy production and desalination. Desalination. 2013, 308 (0):

122-130.

Fig. 4 MDCs were successfully assembled in our project. Desalination rate data were collected

according to conductivity of solution.

Knockout mutants Ctrl

arcA

NPTII

M

Principles of MDC

How does MDC work?

Our MDC device

◆ Electric potential generated by MFC can drive anions and cations being exchanged from the

desalination chamber, by which undrinkable seawater can be desalinated into fresh water.

NAD synthetase

NAD+

e-

nadE

e-

NADH

Microbial Fuel Cells EnableMore Fresh Water and Clean Energy

The arcA knocked out strain produce enhaned electric charge Over-expression of nadE shows better electrogenic performance in E. coli

A reasonable desalination rate was achieved in our MDC device

HUMAN PRACTICE

REFERENCES

CONCLUSION

We thank Profs. Letian Chen, Gang Hao, Yiqun Deng and Drs. Yufei Hu, Yonggang Yang, Bruce

Logan for their helpful suggestion and comments. Our project was supported by

College of Sciences, SCAU; Office of Academic Affairs, SCAU.

ACKNOWLEDGEMENTS

e-

e-

Limiting steps for NADH production vs Our solutions

TCA

ArcA

arcA

◆ Electricity-producing bacteria generate electricity

under anaerobic condition in MFCs. However, TCA

cycle is repressed by ArcA (feedback regulatory reactor

from Arc system) under anaerobic condition, leading to

low metabolic rate and few NADH with releasable

electrons.

TCA

ArcA

arcA◆ Knock-out TCA suppressor arcA gene to

boost up TCA cycle to produce more NADH for

electrons under anaerobic condition.

①e-

e-

e-

e-

e-

e- e-

Anaerobic conditionAnaerobic condition

NAD+

NADH NADH

NAD+

P

P

Principles of a MFC Our MFC device

How does MFC work?

◆ Electricity-producing bacteria (E.coli ) are applied to the anode chamber under anaerobic condition.

Generated electrons are transported from anode to cathode causing eletric current, while the protons are

exchanged via PEM.

Knockout of arcAA

A

B

B

A B

nadE