We characterized existing parts which were designed by the 2013 iGEM team from the National Yang Ming University Taiwan (iGEM13_NYMU-Taipei). The parts that we are adding characterization to are the coding sequence – OxyR, a transcription factor protein (BBa_K1104200) and the regulatory sequence – TrxCp (BBa_K1104201), which is a ROS-induced promoter controlled by OxyR.

We attempted to improve the sensitivity of OxyR to reactive oxygen species (ROS) by increasing the accessible surface area of the reactive cysteine (cysteine-199) on the protein. This cysteine, once oxidized will form a intermolecular disulfide bond with Cys-208, resulting in a conformational change which can activate certain ROS sensitive promoters and therefore positively expressing genes in times of oxidative stress.

Our approach was to use a large set of statistical data to identify certain structural features within the environment of reactive cysteines in a wide variety of protein structures sourced from the Protein Data Bank (PDB). We compared the structural features of these oxidized cysteine environments (the cysteines will form a Sulfenic acid upon oxidation – a post translational modification which is known to be a switch in some proteins) with unmodified cysteine residues within the same proteins. The information we obtained from this large study (of over 300 different proteins) was then used to inform decisions into editing the OxyR gene sequence to mutate the amino acid sequence around the Cys-199. We hoped to make this transcription factor more sensitive to lower concentrations of H2O2 and potentially create a new device which could be used to be a very sensitive indicator of early immune response and infection.

To obtain a comprehensive account of the data used to make the decisions to edit the OxyR nucleotide sequence, please visit our Model page. Our analysis led us to change two amino acids in the OxyR protein surrounding the Cys-199 as seen below.

The Environment surrounding the reactive cysteine-199 of the OxyR protein crystal structure protein (PDB ID: 1i69). Cysteine-199 is colored in Blue and the residues chosen for mutagenesis are highlighted in magenta (Val-147 and Glu-203).

Environment surrounding the reactive cysteine-199 of the OxyR protein crystal structure in reduced form (PDB ID: 1i69) after mutagenesis. New residues (Gly-147 and Ser-203) are colored in light grey.

We mutated amino acids GLN-203 and Val-147 in the original protein. We sent our new sequence to Genscript China to be sequenced, along with the original OxyR sequence. The circuit we used to measure any improvement in our cells is found below:

The OxyR gene is expressed by a constitutive promoter while the inducible promoter TrxC will only express downstream genes (GFP in this case) when it is activated by oxidized form of OxyR, by virture of binding to sites (maked in pink here). When H2O2 levels are high enough the OxyR is activated and GFP is produced. Both constructs were sequenced onto a expression vector pET301(+) and transformed the plasmid into Bl21(DE3) cells. To induce the TrxC promoter we divided four 50ml test tubes of OxyR and OxyR_Mutated into twelve 15ml test tubes, each contained 5ml culture broth and 5ml LB with OD600 0.4. H2O2 was added to 10 of the tubes in the following concentrations:

Tube	Concentration of H2O2added	Final Contents
A1	5mM	5ml A+ 5ml LB+ 5.10μl H2O2
A2	2.5mM	5ml A+ 5ml LB+ 2.25μl H2O2
A3	1mM	5ml A+ 5ml LB+ 1.02μl H2O2
A4	0.1mM	5ml A+ 5ml LB+ 0.10μl H2O2
A5	0.01mM	5ml A+ 5ml LB+ 0.01μl H2O2
A6	0mM (Control)	5ml A+ 5ml LB
B1	5mM	5ml B+ 5ml LB+ 5.10μl H2O2
B2	2.5mM	5ml B+ 5ml LB+ 2.25μl H2O2
B3	1mM	5ml B+ 5ml LB+ 1.02μl H2O2
B4	0.1mM	5ml B+ 5ml LB+ 0.10μl H2O2
B5	0.01mM	5ml B+ 5ml LB+ 0.01μl H2O2
B6	0mM (Control)	5ml B+ 5ml LB

We measured the OD600 and Fluorescence by using plate reader. The data was recorded. After that, we calculated the average OD600 and Fluorescence for all samples. For each of samples, we divided the relative fluorescence value (RFV) by the average OD600. Results are shown below where sample A represents the Original OxyR biobrick (BBa_K1104200) and sample B represents our new OxyR sequence (BBa_K3031018). The blank sample represents BL21(DE3) cells without transformation of plasmid:

Our results here show that our new part including mutations shows no significant difference between the original OxyR part at low H2O2 concentrations. When the concentration of H2O2 increases we see that the GFP signal is less than that of the original OxyR part. Our team believe that our new part is working well but we have realized that the slight conformation changes we likely introduced to the OxyR transcription factor protein may have meant it bind less tightly to the sites upstream of TrxC promoter and therefore expression is reduced. In this case we cannot be sure that our new part is more or less sensitive to oxidation by ROS. We have thought about trying other inducible promoters as they may have slightly different binding sites and our new part could be well suited to one of them. To improve the whole system we now realize taking a strategy to improve the binding of activated OxyR to these inducible promoters would be more logical and a better approach to improve expression of downstream genes. Nonetheless we are very happy we characterized both old and new parts and are still looking forward to trying our mutated OxyR transcription factor with other inducible promoters. We are very happy with our approach taken into trying to improve this part and would like to assist other teams in using structural features around reactive residues to improve parts in the future.