Team:SUIS Shanghai/Results

Result

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.


IGEM   |   WANYUAN   |   SUIS

Website: wanyuan.suis.com.cn

Mail Box: suisigem@outlook.com

No.509, Pingji Rd, Minhang District, Shanghai

Copyright © 2019 Shanghai United International School. All Rights Reserved.