Difference between revisions of "Part:BBa K3490999"
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Unlike BBa_R0010 which includes Lac operator and CAP protein binding site, this part is the core promoter region that only contains -35 to -10 site.
 
Unlike BBa_R0010 which includes Lac operator and CAP protein binding site, this part is the core promoter region that only contains -35 to -10 site.
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<br><b style="font-size:1.5rem">Experiment</b>
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<br>In order to test the function of the T7 promoter, we transformed the plasmid into BL21(DE3). Next, to observe the effects of various IPTG concentrations on NOS expression, we performed SDS-PAGE with different IPTG concentrations. The bacteria is cultured for two hours and induced with IPTG for 12 hours. In Fig. 2 we can observe that the first to the third lane express a similar thin band. Meanwhile, we can see a thicker band in the fourth lane, which means it has a higher protein expression when the IPTG concentration is 1 mM.
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<img src="https://static.igem.org/mediawiki/parts/6/6f/T--NCKU_Tainan--BBa_K3490001-nos-sds_phage.png" style="width:35%;">
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<br>Fig. 2. SDS-PAGE of <i>E.coli</i> BL21(DE3) with different concentrations of IPTG. M: Marker; Lane 1: 0.1 mM IPTG; Lane 2: 0.05 mM IPTG; Lane 3: 0.025 mM IPTG; Lane 4: 1 mM IPTG. The arrow from top to bottom indicates NOS (~40kDa), CsgD (~24kDa), and CsgA (~17kDa).
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<br>As we decided to use an IPTG inducible system, we conducted an experiment to determine whether IPTG concentration and induction time can control the production of nitric oxide. Therefore, we test the IPTG system by using different concentrations and induced at different times. Here, we are using <i>E. coli</i> BL21(DE3) strain. As seen in Fig. 3, the nitric oxide production is both time dependent and IPTG dependent. Thereby, we can observe whether the IPTG inducible system can effectively produce nitric oxide in a certain induction time.
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    <img src="https://2020.igem.org/wiki/images/e/eb/T--NCKU_Tainan--Result-IPTGinduced.png" style="width:50%;">
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  <br>Fig. 3. NOS induced by IPTG with different concentrations at different induced times.
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 14:52, 25 October 2020


Lac promoter without Lac operator and CAP binding site.

Unlike BBa_R0010 which includes Lac operator and CAP protein binding site, this part is the core promoter region that only contains -35 to -10 site.
Experiment
In order to test the function of the T7 promoter, we transformed the plasmid into BL21(DE3). Next, to observe the effects of various IPTG concentrations on NOS expression, we performed SDS-PAGE with different IPTG concentrations. The bacteria is cultured for two hours and induced with IPTG for 12 hours. In Fig. 2 we can observe that the first to the third lane express a similar thin band. Meanwhile, we can see a thicker band in the fourth lane, which means it has a higher protein expression when the IPTG concentration is 1 mM.



Fig. 2. SDS-PAGE of E.coli BL21(DE3) with different concentrations of IPTG. M: Marker; Lane 1: 0.1 mM IPTG; Lane 2: 0.05 mM IPTG; Lane 3: 0.025 mM IPTG; Lane 4: 1 mM IPTG. The arrow from top to bottom indicates NOS (~40kDa), CsgD (~24kDa), and CsgA (~17kDa).


As we decided to use an IPTG inducible system, we conducted an experiment to determine whether IPTG concentration and induction time can control the production of nitric oxide. Therefore, we test the IPTG system by using different concentrations and induced at different times. Here, we are using E. coli BL21(DE3) strain. As seen in Fig. 3, the nitric oxide production is both time dependent and IPTG dependent. Thereby, we can observe whether the IPTG inducible system can effectively produce nitric oxide in a certain induction time.


Fig. 3. NOS induced by IPTG with different concentrations at different induced times.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]