Our Parts

We have designed six basic parts, and four composite parts for our project. These are:

After discussing with Dr Markiv, we concluded that in the week of lab time that we had available, we should attempt to build and transform the two inserts for ComR and the GFP.

In the lab we successfully built and transformed these parts into DH5ɑ E. coli, but unfortunately we did not have the time to test our parts.

As part of our contribution to future iGEM teams and the competition, we have created parts for all of our sequences so they can be used in future.

Separate from the wet lab work, we also designed a two-plasmid system to confer the ability to reduce nitrous oxide in aerobic conditions to E. coli. We believe this is a step above similar systems that have been described in literature, and we hope that our contribution will help future researchers in exploring the possibilities of nitrous oxide bioremediation.

BBa_K5111000 - Binding site for ComR protein

This basic part provides the sequence within the comC promoter region which the ComR protein binds to. The sequence is derived from E. coli K-12 MG1655, as predicted by EcoCyc - bhsA is a synonym for comC.

BBa_K5111001 - ComR coding sequence

The ComR coding sequence in our part is derived from the genome of Top 10 E. coli. The role of ComR is explained fully in our project design, but simply it downregulates the expression of ComC which subsequently increases permeability of the outer membrane to copper. This would theoretically result in higher levels of copper in the bacteria. (note: Mermod M, Magnani D, Solioz M, Stoyanov JV. The copper-inducible ComR (YcfQ) repressor regulates expression of ComC (YcfR), which affects copper permeability of the outer membrane of Escherichia coli. Biometals. 2012 Feb;25(1):33-43. doi: 10.1007/s10534-011-9510-x. Epub 2011 Nov 17. PMID: 22089859. )

Whilst clearly designed around our project, we envision this part in particular as presenting an opportunity for wider usage. Many of the copper-uptake systems already on the registry focus on binding copper for bioremediation, rather than increasing its bioavailability in the organism. Therefore, our ComR system presents an opportunity for other enzymes with copper cofactors, like Cytochrome c oxidase (note: Fontanesi F, Soto IC, Barrientos A. Cytochrome c oxidase biogenesis: new levels of regulation. IUBMB Life. 2008 Sep;60(9):557-68. doi: 10.1002/iub.86. PMID: 18465791; PMCID: PMC2630494. ) and superoxide dismutase-1. (note: Sea K, Sohn SH, Durazo A, Sheng Y, Shaw BF, Cao X, Taylor AB, Whitson LJ, Holloway SP, Hart PJ, Cabelli DE, Gralla EB, Valentine JS. Insights into the role of the unusual disulfide bond in copper-zinc superoxide dismutase. J Biol Chem. 2015 Jan 23;290(4):2405-18. doi: 10.1074/jbc.M114.588798. Epub 2014 Nov 28. PMID: 25433341; PMCID: PMC4303690. )

BBa_K5111002 - Insert containing ComR sequence

Transformation of the ComR gene into E. Coli in order to measure increase in copper uptake has not been undertaken before. Due to lack of time in the lab, we were unable to do this ourselves, but we built the part required to do so.

We have also designed tests which can be used to assess expression and binding to the ComR binding site. These are detailed on the Engineering page.

BBA_ K5111004 - GFP insert for testing ComR binding

This part is designed to test the effectiveness of ComR in downregulating expression. The CDS for BBa_E0040 is downstream of the ComR binding site, so in the presence of ComR, fluorescence should be downregulated. This part should allow semi-quantitative analysis of ComR binding to be undertaken.

BBa_ K5111008 and BBa_ K5111009 - inserts for NosR system

This plasmid design was inspired by the work of the 2021 Wageningen iGEM team, Cattleyst, and Zhang et al. in their article “Functional assembly of nitrous oxide reductase provides insights into copper site maturation”.

We have improved on their designs to provide a new contribution for iGEM in the following ways:

• Zhang et al. did not publish the sequences for their plasmids, and regardless they would likely be incompatible with the biobrick standards. However, they saw promising results for protein stability in aerobic conditions, which informed our choice of species.
• Cattleyst took the sequences from their parts from P. stutzeri JM300. We believe that P. stutzeri ZoBell ATCC 14405 presents a more promising option for reduction in aerobic conditions, as detailed in the project design. Additionally, many of their sequences were not compatible with RFC[10] which we have changed in our sequences.

The two parts contain NosR, other Nos operon proteins, ApbE and tatE. We envision that they could be placed in, for example, the psB1C3 and psB4K5 plasmids which are high-copy, compatible plasmids.

Parts in the first insert

Parts in the second insert

The sequences used are derived from P. stutzeri ZoBell which has shown promising signs of being able to reduce N2O aerobically (note: Wang Z, Vishwanathan N, Kowaliczko S, Ishii S. Clarifying Microbial Nitrous Oxide Reduction under Aerobic Conditions: Tolerant, Intolerant, and Sensitive. Microbiol Spectr. 2023 Mar 16;11(2):e0470922. doi: 10.1128/spectrum.04709-22. Epub ahead of print. PMID: 36926990; PMCID: PMC10100939. ) - a feat not demonstrated by either Zhang et al. or Cattleyst.

We believe that combined, these two parts present a potential design for a two plasmid system which could give E. coli the ability to reduce nitrous oxide. We believe this will be a step forward in bioremediation if achieved, as it would lead to aerobic reduction of N2O in a chassis with a simple system to increase copper uptake.