Iron QS

Table 1: Fluorescence Quantitative (Excitation: 485nm/ Emission: 528nm) and optical dentity (600nm) Measurement of our Iron QS system under Iron rich and iron depleted medium. Culture under each condition is measured with three sample, and average value and standard deviation is calculated.

Blank	Sample1	Sample2	Sample3	AVG	SD
RFV	947652	960382	948677	952237
OD	0.591	0.628	0.593	0.604
RFV/OD	1603472.081	1529270.7	1599792.58	1577511.787	34144.6581
Iron QS+DP				AVG
RFV	391548	401613	398010	398010
OD	0.123	0.117	0.116	0.119
RFV/OD	3183317.073	3432589.74	3431120.69	3349009.169	117163.5394
Iron QS+FeSO4				AVG
RFV	545133	565243	552435	554270
OD	0.341	0.344	0.345	0.343
RFV/OD	1598630.499	1643148.26	1601260.87	1614379.612	20394.17861

Table 2: Summarize of the result of Fluorescence Quantitative (Excitation: 485nm/ Emission: 528nm) and optical dentity (600nm) Measurement of our Iron QS system under Iron rich and iron depleted medium.

	Blank	Iron QS+DP	Iron QS
RFV(AVG)	952237	397057	554270
OD(AVG)	0.604	0.119	0.343
RFV/OD600	1576551.325	3345985.955	1614379.612

Figure 1- Relative fluorescence values of BL21 cell containing ironQS system after cultured in iron-rich and iron-limited media respectively. The Blank strain was set as controls.

Conclusion:

From the figure we can see that There's a significant difference of expression between iron-free and iron-rich environment. BL21-Iron QS has the highest expression of GFP when adding DP-under low concentration of ferric iron. The expression level between BL21-Iron QS in high Fe concentration medium and blank BL21 is similar, indicating that there's no GFP being expressed in both culture. FUR cannot bind to fur box and repress expression when Fe concentration is low is the medium, and vice versa. In our system, GFP can be expressed efficiently when Fe concentration is low.

Reference

1.Teng Chu, Chunshan Ni, Lingzhi Zhang, Qiyao Wang, Jingfan Xiao, Yuanxing Zhang ,and Qin Liu, A quorum sensing-based in vivo expression system and its application in multivalent bacterial vaccine, Microbial Cell Factories, DOI 10.1186/s12934-015-0213-9

SDS-Page

Figure 2: SDS-PAGE result of Iron-QS system expressing ORF 81.The antigen ORF81 with the poly His tag we constructed has a mass of 29 kDa. Lane 1, 2, and 3 are sample from high cell density, and lane 4, 5, 6 are sample from low cell density culture. As the result of SDS-page indicated, all six lanes share similar peptite band but different staining intensity, and there's presence of protein that is near 25 kDa for all six lanes. This pattern suggests that both culture are induced and the difference in staining intensity is a result of different cell density, so it's still not clear whether it's our protein of interest.

Western Blotting

Figure 3: Western blotting result of Iron-QS system expressing ORF 81. Lane 1, 2, and 3 are three repititions of sample A, and lane 4, 5, 6 are three repititions of sample B. As the result of western blotting indicated, three lanes of sample A share the same polypeptite band, so do three lanes of sample B. This suggests a difference in protein expression between sample A and B, which is a result of induction and repression of our system. Iron QS in sample A is ideally expressed as the iron chelator-DP-reduce the ferric iron concentration in the medium. The sytem in sample B is repressed by iron-bound holo FUR. However, three possible bands for protein of interest corresponds to 43 kDa molecular on the ladder. Although there's a difference between the result of western blotting and our ideal protein size (29 kDa), this might be caused by post translational modification of protein. Possible chemical modification, such as glycosylation, methylation, and phosphorylation, may contribute to the variance of protein size. Most membrane-bound proteins expressed in the endoplasmic reticulum are glycosylated, which entail covalent addition of sugar moieties to specific amino acids, to some extent [1]. Because the oligosaccharides could be very large, it's possible the bands are results of glycosylation of our protein of interest.

Figure 4: Example of different types of Glycosylation (From ThermoFisher)

OxyR (Old Part Characterization)

Table1. Fluorescence Quantitative (Excitation: 485nm/ Emission: 528nm) and optical dentity (600nm) Measurement of sample A, with the original OxyR transcription factor (BBa_K1104201) and OxyR induced promoter TrxCp (BBa_K1104201) from iGEM13_NYMU-Taipei under six H2O2 concentration (5mM, 2.5mM, 1mM, 0.1mM, 0.01mM,and 0mM). Culture under each concentration is measured with three sample, and average value and standard deviation is calculated.

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

Table2. Fluorescence Quantitative (Excitation: 485nm/ Emission: 528nm) and optical dentity (600nm) Measurement Blank BL21 culture under six H2O2 concentration (5mM, 2.5mM, 1mM, 0.1mM, 0.01mM,and 0mM). Culture under each concentration is measured with three sample, and average value and standard deviation is calculated.

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

Figure 5: Characterization of OxyR (BBa_K1104200) and TrxCp (BBa_K1104201). Comparision of relative fluorescence value (RFV)/OD600 of Sample A, with the original OxyR transcription factor (BBa_K1104200) from iGEM13_NYMU-Taipeiunder and OxyR induced promoter TrxCp (BBa_K1104201), and Blank, unmodifeid the BL21 E. coli, under six H2O2 concentration. Results indicate a significant difference between Sample A and Blank in all concentratio chosed.

OxyR Mutation (Improvement)

Table 3. Fluorescence Quantitative (Excitation: 485nm/ Emission: 528nm) and optical dentity (600nm) Measurement of sample B, with our modified OxyR (BBa_K3031018) and original promoter TrxCp (BBa_K1104201), under six H2O2 concentration (5mM, 2.5mM, 1mM, 0.1mM, 0.01mM,and 0mM). Culture under each concentration is measured with three sample, and average value and standard deviation is calculated.

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

Table 1: Fluorescence Quantitative (Excitation: 485nm/ Emission: 528nm) and optical dentity (600nm) Measurement of sample A, with the original OxyR transcription factor (BBa_K1104201) and OxyR induced promoter TrxCp (BBa_K1104201) from iGEM13_NYMU-Taipei under six H2O2 concentration (5mM, 2.5mM, 1mM, 0.1mM, 0.01mM,and 0mM). Culture under each concentration is measured with three sample, and average value and standard deviation is calculated.

H2O2 (mM)					Average	SD
5	RFV	360739	360956	342911	354868.6667
	OD600	0.22	0.23	0.19	0.213333333
	RFV/OD	1639722.727	1569373.913	1804794.7	1671297.126	170344
2.5	RFV	484957	476730	475935	479207.3333
	OD600	0.236	0.241	0.247	0.241333333
	RFV/OD	2054902.542	1978132.78	1926862.3	1986632.557	52616.5847
1	RFV	490687	473913	479366	481322
	OD600	0.188	0.212	0.199	0.199666667
	RFV/OD	2610037.234	2235438.679	2408874.3	2418116.762	153068.798
0.1	RFV	379381	380604	331979	363988
	OD600	0.338	0.331	0.336	0.335
	RFV/OD	1122428.994	1149861.027	988032.73	1086774.253	70713.2434
0.01	RFV	370919	361048	347991	359986
	OD600	0.44	0.431	0.436	0.435666667
	RFV/OD	842997.7273	837698.3759	798144.49	826280.1995	20012.2323
0	RFV	253738	243455	241936	246376.3333
	OD600	0.275	0.295	0.289	0.286333333
	RFV/OD	922683.6364	825271.1864	837148.78	861701.2039	43392.8731

Table 2. Fluorescence Quantitative (Excitation: 485nm/ Emission: 528nm) and optical dentity (600nm) Measurement Blank BL21 culture under six H2O2 concentration (5mM, 2.5mM, 1mM, 0.1mM, 0.01mM,and 0mM). Culture under each concentration is measured with three sample, and average value and standard deviation is calculated.

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

Table 3. Blank, control, Fluorescence Quantitative Measurement

Figure 6: Our Improved OxyR (BBa_K3031018) oxidative stress sensitivity test comparing to original OxyR transcription factor (BBa_K1104200) from iGEM13_NYMU-Taipeiunder. Sample A is culture with the original OxyR transcription factor (BBa_K1104200) from iGEM13_NYMU-Taipeiunder and OxyR induced promoter TrxCp (BBa_K1104201); Sample B is replaced with our modified OxyR (BBa_K3031018) and Blank is just unmodifeid BL21 E. coli, under six H2O2 concentration. Results indicate a significant difference of relative fluorescence value (RFV)/OD600 between Sample A and Sample B in all concentrations tested, suggesting our modified OxyR does not show any significant improvement in the sensitivity toward H2O2 and has a decreased expression at higher concnetrations.

Future Direction

Since we do not have time to finish wet lab testing on cell wall anchoring system before Giant Jamboree with all finished design and ordered plasmid, we will finish the experiment and collect quantitative data in the future. Also, we may test the effect of out engineered system on living koi fish with a way without any ethical issue.

References:

Wormald, Mark R., Petrescu, Andrei J., Pao, Ya-Lan, Glithero, Ann, Elliott, Tim, & Dwek, Raymond A. (2002). Conformational studies of oligosaccharides and glycopeptides: Complementarity of NMR, X-ray crystallography, and molecular modelling.(Glycobiology). Chemical Reviews,102(2), 371-386.