Difference between revisions of "Part:BBa K3490003"

 
 
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This year, our team aims to reduce intraocular pressure (IOP) through the contact lens with engineered <i>E. coli</i> that can produce Nitric Oxide (NO). We aimed to make our plasmid express NOS and support our bacteria to bind to the contact lenses. Since <i>E.coli</i> doesn't produce nitric oxide, we ordered a sequence containing a lacO-T7 promoter, B0034 RBS, and bsNOS. Then, we ligate the sequence by inserting it into the PUC plasmid and combining it with another sequence (<i>pLac</i>, RBS, <i>csgA</i>, RBS, <i>csgD</i>, <i>LacI</i>) from IDT and transformed into <i>E. coli</i> DH5-Alpha. By doing so, NOS can convert L-arginine into nitric oxide, thus releasing nitric oxide in the eyes.<sup>[1]</sup>
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To ensure the plasmid contains those two sequences, we conduct PCR with each sequence’s primer separately. Fig.1 shows that the plasmid contains both of the desired sequences.
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<p align="center">Fig. 1. Confirmation of our construction by PCR. M: Marker; Lane 1: <i>nos</i> (~2400 bp); Lane 2: <i>csgA</i>-<i>csgD</i> (~1500 bp).</p>
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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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<p align="center">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). </p>
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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/d/d3/T--NCKU_Tainan--results-iptg2.png" style="width:50%;">
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  <p align="center">Fig. 3. NOS induced by IPTG with different concentrations at different induced times.</p>
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 22:45, 27 October 2020


IPTG inducible NOS

This year, our team aims to reduce intraocular pressure (IOP) through the contact lens with engineered E. coli that can produce Nitric Oxide (NO). We aimed to make our plasmid express NOS and support our bacteria to bind to the contact lenses. Since E.coli doesn't produce nitric oxide, we ordered a sequence containing a lacO-T7 promoter, B0034 RBS, and bsNOS. Then, we ligate the sequence by inserting it into the PUC plasmid and combining it with another sequence (pLac, RBS, csgA, RBS, csgD, LacI) from IDT and transformed into E. coli DH5-Alpha. By doing so, NOS can convert L-arginine into nitric oxide, thus releasing nitric oxide in the eyes.[1]

To ensure the plasmid contains those two sequences, we conduct PCR with each sequence’s primer separately. Fig.1 shows that the plasmid contains both of the desired sequences.

Fig. 1. Confirmation of our construction by PCR. M: Marker; Lane 1: nos (~2400 bp); Lane 2: csgA-csgD (~1500 bp).



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
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 229
    Illegal SapI.rc site found at 547