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.

OxyR is activated by ROS (Reactive Oxygen Species) which contains a reactive cysteine residue at position 199 (Cys-199). Upon exposure to a ROS, Cys-199 is oxidized resulting in the formation of an intermolecular disulfide bond between Cys-199 and Cys-208. This causes a conformational change, activating the OxyR transcription factor which can bind to regularly sequences and thus positively regulate some genes.

TrcC promoter is a regulatory sequence for the thioredoxin (TrxC) gene. It contains two Transcription Factor Binding Sites located upstream for OxyR to positively active expression. The OxyR gene then is said to be a positive regulatory gene for the oxidative response as it controls the activation of promoters for the expression of proteins which are induced by treatment with H2O2.

These promoters were used by iGEM13_NYMU-Taipei as a means for detecting infection of bees by the fungus Nosema ceranae. One such promoter is the TrxCp (BBa_K1104201). They also created the part (BBa_K1104241) consiting of the TrxC promoter and the GFP generator (BBa_E0840). These parts have applications for our project as fish are known to produce H2O2 in response to infection by pathogens also. We added our experimental data to each of these parts mentioned above.

This construct will express OxyR all the time but downstream genes from TrxC promoter will only be expressed upon binding of active OxyR to the binding regions. OxyR is activated by teh oxidation of a reactive cysteien resiude (Cys-199) by a reactive oxygen species (ROS).

For our characterization we synthesized the circuit used by iGEM13_NYMU-Taipei containing a constitutive promoter expressing OxyR upstream of the TrxC promoter and OxyR binding sites. GFP reporter gene was inserted downstream of the TrxC promoter and thus was said to be under control of ROS. This construct was transformed into Bl21 (DE3) E.coli cells and we performed an assay which measured GFP florescence with treatment with various concentrations of H2O2.

H2O2 (mM)					Average	SD
5	RFV	567236	575491	568431	570386
	OD600	0.22	0.24	0.221	0.227
	RFV/OD	2578345.45	2397879.1	2572085.97	2516103.53	83636.2982
2.5	RFV	583944	586687	580442	583691
	OD600	0.195	0.212	0.205	0.204
	RFV/OD	2994584.61	2767391.5	2831424.39	2864466.83	95648.7650
1	RFV	945200	975187	954601	958329.333
	OD600	0.31	0.312	0.323	0.315
	RFV/OD	3049032.25	3125599.3	2955421.05	3043350.89	69591.0552
0.1	RFV	450025	449077	448871	449324.333
	OD600	0.302	0.299	0.294	0.29833333
	RFV/OD	1490149.00	1501929.7	1526772.10	1506283.62	15264.9923
0.01	RFV	303234	307976	308938	306716
	OD600	0.341	0.338	0.338	0.339
	RFV/OD	889249.266	911171.59	914017.751	904812.872	11066.3002
0	RFV	180294	183250	183005	182183
	OD600	0.183	0.184	0.19	0.18566666
	RFV/OD	985213.114	995923.91	963184.210	981440.412	13629.5509

H2O2 (mM)		Average	SD
5	RFV	488242
	OD600	0.648
	RFV/OD	753459.8765	49720
2.5	RFV	456371
	OD600	0.585
	RFV/OD	780121.3675	38567
1	RFV	499338.5
	OD600	0.612
	RFV/OD	815912.5817	30909
0.1	RFV	481699
	OD600	0.573
	RFV/OD	840661.4311	36491
0.01	RFV	468741
	OD600	0.586
	RFV/OD	799899.3174	49183
0	RFV	473121.5
	OD600	0.599
	RFV/OD	789852.2538	67900

This is the relative fluorescence value (RFV)/OD600, an indication of promoter performance, of sample A and a control group under different H2O2 concentration. Similar to the trend of the original result from team NYMU-Taipei, the RFV/OD600 value tend to increase as the concentration of H2O2 increase, despite the gradual decreasing fluorescence value after the concentration exceed 1mM. Accelerating protein degradation of GFP might be a possible explanation for this distinction. Protein degradabilities is said to increase along with peroxide concenration.

References:

Cheolju Lee, Soon Mi Lee, Partha Mukhopadhyay, Seung Jun Kim, Sang Chul Lee, Woo-Sung Ahn, . . . Seong Eon Ryu. (2004). Redox regulation of OxyR requires specific disulfide bond formation involving a rapid kinetic reaction path. Nature Structural & Molecular Biology,11(12), 1179-85.

Fredrik Åslund, Ming Zheng, Jon Beckwith, & Gisela Storz. (1999). Regulation of the OxyR transcription factor by hydrogen peroxide and the cellular thiol—disulfide status. Proceedings of the National Academy of Sciences of the United States of America,96(11), 6161-6165.