Team:NCKU Tainan/Design

Design

Sensing

Nitrate and nitrite concentration is an important index in aqua quality, so we decided to monitor the nitrate and nitrite concentration in the water. We used the biobrick K381001 from BCCS-Bristol 2010 iGEM team to do the nitrate and nitrite sensing. The biobrick K381001 is composed of a nitrate-sensing promoter PyeaR, a strong RBS B0030 and a reporter gene GFP. The promoter PyeaR isn’t repressed under aerobic conditions, but it’s inhibited by protein NsrR, which is native to most E. coli . When nitrate or nitric oxide is bound to NsrR, the inhibition is halted. On the other hand, PyeaR is mainly activated by phospho-NarL, and NarL is phosphorylated by nitrate or nitrite (Lin et al ,2007). Accordingly, we can use K381001 as a sensor to detect nitrate, whose concentration can be estimated by quantified the fluorescence level.

Besides, in order to enhance the nitrate-sensing accuracy, we also planned to modify the biobrick K381001. By adding an additional nsrR binding sequence to K381001, the fluorescence basal level can be decreased since the additional nsrR binding sequence can reinforce the repression to PyeaR under no nitrate condition. As a result, the interference to nitrate-detection can be decreased as much as possible, and sensing of nitrate can be more accurate.

Regulation

In aquaculture industry, sickness or massive death of aquatic products may be attributed to virus, temperature and so on. However, increase of nitrate is also a serious problem. High concentration of nitrate may cause not only sickness of aquatic products but also eutrophication which leads to low dissolved oxygen in water body, and then destroys the water ecosystem. In addition, the discharge of nitrate-rich waste water can have negative influence on water ecosystem. To sum up, high amount of nitrate can lead to aquaculturing, environmental and ecological problem.

Due to the problem above, we aim at reducing the nitrate ion in the aquaculture water body. Moreover, we expect metabolite could be some valuable products. After literature searching, we didn’t choose the denitrification process which involves lots of enzymes and toxic intermediate, the nitrate and nitrite respiration [1] and nitrogen assimilation pathway in E. coli [2] fit our goal instead.

According to the literature on nitrate and nitrite respiration, we decided to take advantage of nitrate reductase (NaR) and nitrite reductase (NiR) to convert nitrate to ammonia. Surprisingly, both pathways can be found in E. coli; hence, we take E. coli as the primary host. In nitrogen assimilation pathway, ammonium can be converted to glutamate by glutamate dehydrogenase (GDH), and further to glutamine by glutamine synthetase (GS). In these pathways, intermediates of the metabolic pathway are not toxic to the host, E. coli. Moreover, nitrate ion can be successfully transformed into glutamine, an important and valuable amino acid.

Nitrate Assimilation Process

From nitrate to nitrite

In E. coli, there are two ways to catalyze the nitrate to ammonium. One is in the periplasm and the other is in the cytoplasm. Based on our project, we want to capture the nitrate in the waters and change it to other product inside the cytoplasm. As a result, we use the nitrate and nitrite reductase to convert the nitrate to ammonia. At first, we want to use the nar cluster in Pseudonomas, because the activity is much more than the E. coli. We divided the nar cluster into three parts and took it for IDT synthesis. However, due to too high GC content of one-part sequence, it failed to synthesize. Therefore, we are looking for another solution, in which original nar cluster existed in the wild type E. coli MG1655 had been used in our project.

Figure 1. The arrangement of nar cluster in the genome

From nitrite to ammonia

Furthermore, we take advantage of the gene nirBD which encodes the sequence of nitrite reductase to convert nitrite into ammonia. Both two gene are NADH-dependent. The reaction formula shows below:

nitrite + 3 NADH + 5 H+ → ammonium + 3 NAD+ + 2 H2O

According to the data from Brenda database, the specific activity of nitrite reductase is just 0.052 µmol/min/mg in E. coli. We want to amplify the amount of nitrite reductase in the cytoplasm. As a result, we designed the primers as to get the nir cluster gene from genome. The primers contain the restriction sites of HindIII at the 5’ terminal and SpeI at the 3’ terminal. We digested the gene with the two restriction enzymes and then ligased into the pSB1C3 vector. Using the strong promoter PLacI and high copy number ori pUC rather than pMB1. After constructing the plasmid, we transform it into E. coli DH5α for construction. Further, transform the plasmid into E. coli MG1655 for protein expression.

Figure 2. The genetic circuit of BBa_K2275007

Do our biobrick work?

We used the method of griess reaction to test if our biobrick can successfully work. When the broth contained nitrite, the color will turn pink and have absorbance at wavelength 545 nm

Ammonium Assimilation

From ammonium to glutamate

In order to convert ammonium to glutamate, we go through the GDH process and with the gene gudB which encodes the sequence of glutamate dehydrogenase. The gene comes from Bacillus subtilis and the activity is almost twice higher than the gene gdhA which has the same effect in E. coli. The reaction formula is shown below:

ammonium + 2-oxoglutarate + NADPH + H+ ↔ L-glutamate + NADP+ + H2O

Hence, we designed primers which contained the restriction sites of BamHIat 5’ terminal and PstI at 3’ terminal to get the gene from genome. We digested the gene with the two restriction enzymes and then ligased into the pSB1C3 vector which is the same as before. After constructing the plasmid, we transformed it into E. coli DH5α for construction. Further, we transformed the plasmid into E. coli MG1655 for protein expression.

Figure 3. The genetic circuit of BBa_K2275008

From glutamate to glutamine

Next, to convert the glutamate to glutamine, we used the GS pathway. Using the gene glnA which encodes the sequence of glutamine synthetase is needed. The gene comes from Pseudomonas putida. The primers we use to amplify glnA gene contain restriction sites of HindIII and PstI. We digested the gene with the two restriction enzymes and then ligased into the pSB1C3 vector which is the same as before. After constructing the plasmid, we transformed it into E. coli DH5α for construction. Afterward, transformed the plasmid into E. coli MG1655 for protein expression.

Figure 4. The genetic circuit of BBa_K2275009

From ammonium to glutamine

We also constructed the gudB and glnA into the same backbone. Re-design the primers of gudB to reverse the 5’ and 3’ direction compare to previous one. The primers contained restriction sites of XbaI and PstI. Afterward, digested the rev-gudB with XbaI and PstI and BBa_K2275009 with SpeI and PstI. Ligased and transformed into DH5α for construction. Further, transforming into E. coli MG1655 for protein expression.

Figure 5. The genetic circuit of BBa_K2275010

Do our biobrick function?

For the purpose of testing Biobricks function, we use two kit to detect the concentration of glutamate and glutamine; one is Glutamate Colorimetric Assay Kit for analyzing concentration of glutamate, and the other is Glutamine Colorimetric Assay Kit for analyzing concentration of glutamine.

Reference

Sensing

Lin, H. Y., Bledsoe, P. J., & Stewart, V. (2007). "Activation of yeaR-yoaG operon transcription by the nitrate-responsive regulator NarL is independent of oxygen-responsive regulator Fnr in Escherichia coli K-12." Journal of bacteriology, 189(21), 7539-7548.

Regulation

[1] Steen, A., Ütkür, F. Ö., Borrero-de Acuña, J. M., Bunk, B., Roselius, L., Bühler, B., Jahn, D. & Schobert, M. (2013). "Construction and characterization of nitrate and nitrite respiring Pseudomonas putida KT2440 strains for anoxic biotechnical applications." Journal of biotechnology, 163(2), 155-165.

[2] van Heeswijk, W. C., Westerhoff, H. V., & Boogerd, F. C. (2013). "Nitrogen assimilation in Escherichia coli: putting molecular data into a systems perspective." Microbiology and Molecular Biology Reviews, 77(4), 628-695.

[3] Lundberg, J. O., Weitzberg, E., Cole, J. A., & Benjamin, N. (2004). "Nitrate, bacteria and human health." Nature Reviews Microbiology, 2(7), 593-602.