Table 1 shows an overview on the composite parts designed by our team.
Table 1: Parts overview.
|Part:BBa_K3762005||Composite||Hydrogen sulfide sensor to be used with a sulfide oxidising component, with CreiLov reporter.||985|
|Part:BBa_K3762006||Composite||Hydrogen sulfide sensor to be used with a sulfide oxidising component, with CreiLov reporter.||985|
|Part:BBa_K3762007||Composite||Hydrogen sulfide sensor to be used with a sulfide oxidising component, with GFP reporter.||1342|
|Part:BBa_K3762008||Composite||Whole-cell hydrogen sulfide sensor, with CreiLov reporter.||2492|
All our composite parts were designed to be used as hydrogen sulfide sensors. Parts BBa_K3762005, BBa_K3762006, and BBa_K3762007 are sensors for reactive sulfur species and must therefore be used in conjunction with a component that oxidises sulfide to S0, whilst Part BBa_K3762008 is a sensor for sulfide.
All the composite parts were ordered fully synthesized from IDT as a linear sequence, and subsequently cloned into a pENZ004 plasmid vector using Gibson assembly, and transformed into E. coli DH5-alpha cells.
Both part designs take advantage of the characteristics of the repressor protein SqrR. SqrR is a repressor protein that is involved in sulfide-dependent gene expression in the bacterium Rhodobacter capsulatus. When no sulfide is present, SqrR binds to the Psqr promoter, inhibiting the expression of the gene encoding Sulfide:quinone oxidoreductase (SQR). SQR is an enzyme that oxidises sulfide to zero-valent sulfur (S0), which can then conjugate with multiple different nucleophiles or low molecular weight thiols to form reactive sulfur species (RSS) . Upon reaction with a RSS, a di-, tri-, or tetrasulfide bond forms between two cysteine residues of SqrR, resulting in a conformational change that reduces its DNA binding affinity. RSS thereby functions as inducers of the genes under control of the Psqr promoter that is repressed by SqrR.
Hydrogen sulfide sensors to be used with a sulfide oxidising component
Parts BBa_K3762005, BBa_K3762006, and BBa_K3762007 take advantage of the sulfide-binding properties of SqrR, as well as its repressor function, so as to function as a sensor for hydrogen sulfide. When RSS are present, reaction with SqrR induces the expression of the reporter gene. However, in order to function as a hydrogen sulfide sensor, the part must be used in conjugation with a component that oxidises sulfide to S0, such as SQR. In our project, oxidised glutathione (GSSG) was reacted with sulfide to form glutathione persulfide (GSSH), a RSS. This is summarized in figure 1.
Figure 1: Method of hydrogen sulfide detection by parts BBa_K3762005, BBa_K3762006, and BBa_K3762007.
The parts consist of a fluorescent reporter gene placed under control of the Psqr promoter, sqrR, the gene that encodes SqrR, placed under control of a constitutive promoter, as well as biobrick ribosomal binding sites and terminators. Figure 2 shows the components of the composite parts.
Figure 2: Components of composite parts BBa_K3762005, BBa_K3762006, and BBa_K3762007. Figure designed using Pigeon .
The main difference between the three parts is that in Parts BBa_K3762005 and BBa_K3762006, the reporter gene is the fluorescent protein CreiLov, whilst in part BBa_K3762007 the reporter gene is GFP. The relative strength of the constitutive promoters used for sqrR expression also varies, with the promoter used for BBa_K3762005 being relatively stronger.
This was done because we wanted to test wherever the expression level of SqrR affected the sensitivity of the parts.
Part BBa_K3762008 is a whole-cell hydrogen sulfide sensor
Part BBa_K3762008 takes advantage of the sulfide-binding properties of SqrR, as well as its repressor function, so as to function as a sensor for hydrogen sulfide. The part consists of the fluorescent reporter gene CreiLov placed under control of the Psqr promoter, sqr, the gene that encodes SQR, placed under control of a constitutive promoter, sqrR, the gene that encodes SqrR, placed under control of a constitutive promoter, as well as biobrick ribosomal binding sites and terminators. The components of the composite part are shown in figure 3.
Figure 3: Components of composite part BBa_K3762008. Figure designed using Pigeon .
When hydrogen sulfide diffuses across the cell membrane, the constitutively expressed SQR oxidises sulfide to S0. The oxidized S0 can then react with SqrR, inducing the expression of the reporter protein. This is illustrated in figure 4.
Figure 4: Method of hydrogen sulfide detection by part BBa_K3762008.
 Shimizu, T., Shen, J., Fang, M., Zhang, Y., Hori, K., Trinidad, J. C., Bauer, C. E., Giedroc, D. P., & Masuda, S. (2017). Sulfide-responsive transcriptional repressor SqrR functions as a master regulator of sulfide-dependent photosynthesis. Proceedings of the National Academy of Sciences of the United States of America, 114(9), 2355–2360. https://doi.org/10.1073/pnas.1614133114
 Bhatia, S. & Densmore, D. Pigeon: a design visualizer for synthetic biology. ACS Synth. Biol. 2, 348–350 (2013).