Team:Kent/Project

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No to NO: A novel approach to reduce greenhouse gas

Abstract

In today’s rapidly changing environment greenhouse gases such as NO are an issue that need to be addressed. NO has been proven to have a detrimental impact on the environment and iGem Team Kent 2013 will provide a solution that focuses on reducing the amount of NO formed in waste water. Our system will utilise an engineered strain of E. coli which will be capable of converting this excess NO into ammonia. Our Biobricks have been designed to enable the detection of NO using the norV promoter. The NO can then be converted into ammonia via the nitrite reductase enzyme encoded by the E. coli gene nrfA. Our solution will have many advantages over the current approaches to waste water treatment such as reduced cost and risk of contamination. Our system will provide a source of recycled ammonia and could be a greener alternative to the Haber Bosch process.

Introduction

Background of our problem

Nitric oxide is a greenhouse gas that is evolved during the processing of wastewater. Oxides of nitrogen (NOX) are key oxidants that have a role in the photochemical production of ozone, whilst also being linked with an increase in the oxidising capacity of the atmosphere2. Too much NOX within the atmosphere has a detrimental effect on these processes, adding to the greenhouse effect and causing acid rain2.

Strategy

Team Kent aims to tackle the production of nitric oxide from waste water treatment processes by exploiting systems already present in laboratory strains of E. coli: NorR, NorVW and nrfA. Our idea is to engineer a plasmid to that will express the nitrite reductase enzyme nrfA in an NO-dependent manner under the control of the norV promoter: in the presence of NO, nrfA will be expressed and will convert the NO to ammonia.

NrfA

The nitrite reductase nrfA is encoded within the nrf operon that is found in enteric bacteria such as E. coli. The primary function of nrfA is to enable the use of nitrite as a respiratory electron acceptor, forming ammonia as a final product. In addition, NO is a catalytic intermediate in this reaction, and can be can then also be used as a substrate reduction to ammonia by nrfA 3.

NorR

NorR is the transcriptional regulator of the norVW operon encoding NO-detoxifying flavorubredoxin and associated oxidoreductase in E. coli. It is a nitric oxide (NO) sensing bEBP that is σ54 – dependent, and forms as an oligomer wrapping the promoter DNA around itself. Additionally, it is made of 3 domains, GAF, AAA+ and HTH. The regulatory GAF domain represses AAA+ activity, but NO binding to GAF relieves this repression. AAA+ contacts σ54 and induces open promoter complex formation powered by

N-terminal regulatory GAF domain contains non-haem iron centre that binds NO. It represses AAA+ domain ATPase activity in the absence of NO, but NO binding relieves this repression. AAA+ domain is the active component of transcriptional activation. When ATP is bound, it contacts σ54 via a loop containing a conserved GAFTGA motif. On phosphate release following an ATP hydrolysis cycle, it relocates the σ54 RNA polymerase holoenzyme to induce open promoter complex formation

HTH (helix-turn-helix) domain binds to 3 enhancer DNA sequences. All 3 are required for formation of NorR oligomer at promoter, and thus AAA+ domain ATPase activity. DNA wrapping at this type of promoter is assisted by integration host factor (IHF) in E. coli. In contrast to many other bEBPs, in which the regulatory signal stimulates oligomerization, NorR oligomerization precedes NO signal sensing. By preforming at the norVW promoter, NorR is ‘primed’ to rapidly initiate norVW transcription in response to an NO signal

Existing technology: limitations and scope for improvement

NorR is the transcriptional regulator of the norVW operon encoding NO-detoxifying flavorubredoxin and associated oxidoreductase in E.coli. It is a nitric oxide (NO) sensing bEBP that is σ54 – dependent, and forms as an oligomer wrapping the promoter DNA around itself. Additionally, it is made of 3 domains, GAF, AAA+ and HTH. The regulatory GAF domain represses AAA+ activity, but NO binding to GAF relieves this repression. AAA+ contacts σ54 and induces open promoter complex formation powered by ATP hydrolysis.4

N-terminal regulatory GAF domain contains non-haem iron centre that binds NO. It represses AAA+ domain ATPase activity in the absence of NO, but NO binding relieves this repression. AAA+ domain is the active component of transcriptional activation. When ATP is bound, it contacts σ54 via a loop containing a conserved GAFTGA motif. On phosphate release following an ATP hydrolysis cycle, it relocates the σ54 RNA polymerase holoenzyme to induce open promoter complex formation.4, 5

HTH (helix-turn-helix) domain binds to 3 enhancer DNA sequences.2 All 3 are required for formation of NorR oligomer at promoter, and thus AAA+ domain ATPase activity. DNA wrapping at this type of promoter is assisted by integration host factor (IHF) in E.coli. In contrast to many other bEBPs, in which the regulatory signal stimulates oligomerization, NorR oligomerization precedes NO signal sensing. By preforming at the norVW promoter, NorR is ‘primed’ to rapidly initiate norVW transcription in response to an NO signal.4, 5

References

1. Cristina Muñoz, C., Paulino, L., Monreal, C., Zagal, E., Chilean Journal of Agricultural Research 70, 3: 485-497

2. Fowler, D., Coyle, M. et al, The Global Nitrogen Cycle in the twenty-first century (2013) Philosophical Transactions of the Royal Biological Society 368 no. 1621

3. Clarke, TA., Mills, PC. et al (2008). Escherichia coli cytochrome c nitrite reductase NItalic textrfA.. Methods in Enzymology. 437: 63-77. * Bush M, Ghosh T, Tucker N, Zhang X, Dixon R: Transcriptional regulation by the dedicated NO sensor, NorR: a route towards NO detoxification; Biochem. Soc. Trans. (2011) 39: 289–293; doi:10.1042/BST0390289

4. Bush M, Ghosh T, Tucker N, Zhang X, Dixon R: Transcriptional regulation by the dedicated NO sensor, NorR: a route towards NO detoxification; Biochem. Soc. Trans. (2011) 39: 289–293; doi:10.1042/BST0390289

5. Tucker N, Ghosh T, Bush M, Zhang X, Dixon R: Essential roles of here enhancer sites in σ54 – dependent transcription by the nitric oxide sensing regulatory protein NorR; Nucl. Acids Res. (2010) 38(4): 1182 – 1194; doi: 10.1093/nar/gkp1065

Materials, Methods and Results of the Project

Discussion

Throughout our 18-week summer project we collected a number of different results that enabled us to collate and draw conclusions with. From a number of different transformation plates, gels and ligations; we were successfully able to produce our BioBricks NorV and nrfA.

Colony PCR

Through the use of colony PCR we were able to amplify our required BioBricks out of the genomic DNA of E. coli. As can be seen in experiment 3, with the use of our primers we designed through using various software, we were successful in producing the correct size fragments for our BioBricks. As we used colony PCR there is a chance that what was amplified had other sections of DNA elongated with the use of Taq polymerase through non-specific binding or other sequences of DNA being complementary to our oligonucleotide. However the likelihood of this was so small that we decided to continue as we had repeated the experiment numerous times.

Agarose gels

After designing our primers and knowing the base pair lengths of our insert, we were able to use agarose gels as seen in experiment 4 after PCR purification in order to see if they were the right length (Figure 6). In order to estimate if they were the right length, we used DNA marker bands that were standardised, showing us fragment lengths along the gel. This allowed us to visualise and estimate whether or not we had our insert. The use of agarose gels was quick and efficient, allowing us to carry on with other experiments when we got positive results. There are however margins of error with agarose gels, as they do not show differences in small amounts of fragments, so we sometimes had to go on the assumption that the enzymes had cut what they should have, with fragments being only 6 base pairs sometimes. We did however do positive controls on each gel to ensure the enzymes were working correctly in order to reduce this margin of error.

Ligations

The majority of our work over the summer period was spent on trying to get ligations to work. We had troubles using both rapid ligation kits and normal ligation kits. Through using a number of diagnostic gels, we were able to deduce that the rapid ligation kit was unsuitable for use and we switched to using a normal ligation kit, and with varying amounts of plasmid : insert ratios (norV and nrfA) (1:0, 1:1, 1:3 and 1:5) and a number of contaminated suspensions, we were able to achieve correct ligations of our single BioBricks at the last minute! We ran a ligation trying to stick together both BioBricks into the plasmid, however that ligation did not work so we were unable to test the products in the way which we initially wanted to (Figures 5 and 7).

Future Experiments

As we had ran out of time due to having problems with ligations, we were unable to complete the joining of both of our BioBricks together, and unable to utilise the GFP we had prepared for ligations. If we had more time, we would have ligated both norV and nrfA together to complete our original function of using nitric oxide to produce ammonia. We would also have joined GFP to NorV in order to receive visual information that our promotor was working efficiently. The activation of NorV would have increased the production of GFP allowing us to image it in real-time.

We ideally would have liked to use western blotting in order to ensure that the protein made by our product was correct. This would have allowed us to double check that the insert was working correctly and ensure reliability and validity. We would have first had to have transformed the plasmid in to bacteria to have been able to do this however, and we did not have enough time. As stated earlier, we were only able to take an educated guess that the inserts were at about the right length with 0.75% agarose gels.

We would have also liked to gain some quantitative data on the amount of nitric oxide that was able to be converted into ammonia with this system, and the precise biochemical reactions involved. Knowing how many moles of cofactors and enzymes are required in each individual E. coli before they become detrimental would have been useful. This would have allowed us to know the maximum yield without being wasting resources. This would also have told us whether increasing this innate process in E. coli would kill it. If this were the issue we would try to resolve it by balancing out other systems already in place in order to ensure the smooth running of the process, possibly by creating other BioBricks. We would have been able to perform these experiments if the ligation of the two BioBricks together had worked.

As our project has been very focussed on its applications, we are well aware that there are different microenvironments in different waste water treatment processes. For more future experiments, we were planning on taking samples from different waste water treatment plants to see the conditions and find an organism that perhaps would be more suited to it than our model E. coli organism is. We then would have transformed the plasmid into said organism to see if the plasmid was compatible with the organism and how efficient it was.

GFP = green fluorescence protein. PCR = polymerase chain reaction.