Team:SZ-SHD/Contribution

Contribution

Team contribution

We evaluated the part (BBa_K518010), using the plasmid circuit pss1. Since the effect of irradiation time yet investigated previously, we designed the experiment to evaluate the response of SulAp to different UV radiation time (see methods and protocols).


The result of eGFP expression after irradiated by UVC, unit: fluorescence per OD. eGFP-: non-recombinant BL21 irradiated under UVC (without pSS1); eGFP- UV-: non-recombinant not exposed under UV. 0min: recombinant BL21, no UV exposure; 0.5min: recombinant BL21 irradiated under UV for 30 seconds; 1min: recombinant BL21 irradiated under UV for 1 min; 2min: the recombinant BL21 irradiated under UV for 2 min.

Our result suggested that the expression level of gene behind SulAp increases as induction time (UV exposure time) increases. Which the 2 min exposure could result in a maximum expression level ~2 times greater than exposed for 30 seconds.

We have also studied the viability of applying T4 lytic proteins as a suicide switch to indue the cellular disruption. (Parts: BBa_K112000; BBa_K112012, submitted by UC Berkeley iGEM team in 2008.), under the regulation of pBAD promoter (BBa_K808000). Protocols

The figure shows a decline in cell count of recombined E. coli (strain DH10B) after induced by 1mM arabinose when OD600 ~0.6. Comparing to the non-transformed stain (control), the cell number of recombined strain (pSB1C3-pBAD-T4) significantly lowered during the 30 hours after induction.

We conclude that the pBAD promoter could be used to induce the expression of lytic proteins and result in cell death. Further experiments also revealed that T4 lysis releases cytoplasmic proteins.

the release of xncht after induction by arabinose (1mM) was measured over 42 hours: the abundancy of protein released ≈ the concentration of proteins in supernatant of induced bacterial culture minus uninduced. Where the concentration could be determined by measuring the absorption of light with 280nm wavelength. Results have a PMCC value of 0.

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