Difference between revisions of "Part:BBa K3156000"

 
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===Usage and Biology===
 
===Usage and Biology===
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<h3 id="CBD">Description</h3>
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<p>In our design, the induction signal will be detected and stored in the plasmid DNA sequence of our genetically modified E. coli. The bacteria will be gathered from the capsule after it left human body and sent to lab for further quantitative analysis in order to represent the inflammation level in colon. Therefore, in our project, the fluorescence intensity will be the index that represents levels of gut inflammation. <br>Since then, we first needed to develop a standard quantitative measurement protocol, so we studied the following articles: Zong, Y et al (2017)[1] and Zhang, H. M. et al(2015). </p>
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<p style="text-align:center;"><img src="https://2019.igem.org/wiki/images/7/7f/T--SHSBNU_China---gfp_zqd.png" width="800" height="100"/>
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<b>Figure 1.We constructed our reporter circuit with sfgfp and pTac promoter</b>
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<p>Following reference, we determined the experimental procedure:<br>
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Flat-bottom 96-well plates and sealing film were used throughout the study. Bacteria harboring parts/circuits of interest were inoculated from plates to LB medium and grown overnight (8−12 h, 1000 rpm, 37 °C, mB100-40 Thermo Shaker). Ten microliters of each overnight culture was sequentially diluted into 130 μL of fresh medium twice; the total dilution fold was 196. After growing the diluted cultures for ∼3 h, we diluted the exponentially growing cultures 700-fold using fresh medium; the dilution process was as follows: 10 μL of cell culture is added to 130 μL of M9 medium, which is followed by diluting 3 μL of this into 147 μL. Then, cultivation continued (1000 rpm, 37 °C, mB100-40); atspecifictimepoints,a2−50μL aliquot of each culture was transferred to a new plate containing 200 μL of PBS with 2 mg/mL kanamycin preadded to terminate protein expression. For the time course of cell growth after 700-fold dilution, OD 600 was recorded using Varioskan Flash (Thermal Scientific); the time interval was 5 min.
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<figure>
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<p style="text-align:center;"><img src="https://2019.igem.org/wiki/images/4/49/T--SHSBNU_China---characterization-step.jpeg" width="800" height="200"/>
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<figcaption>
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<b>Figure 2.Experimental procedure for recording the time course of gene expression using flow cytometry.</b>
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</figcaption>
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</figure>
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<p>The graph below shows our result.</p>
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<figure>
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<p style="text-align:center;"><img src="https://2019.igem.org/wiki/images/e/e5/T--SHSBNU_China---Ptac-result.png" width = "900" height ="400"/>
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<figcaption>
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<b>Figure 1.Ptac response curve in medium copy number vector.</b>
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</figcaption>
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</figure>
  
 
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Revision as of 19:12, 21 October 2019


pLac Promoter - sfGFP

This part consists of a inducible pLac promoter and a sfGFP coding sequence which can be used for quantitative measurement of base-editing efficiency.


Description

<body>

In our design, the induction signal will be detected and stored in the plasmid DNA sequence of our genetically modified E. coli. The bacteria will be gathered from the capsule after it left human body and sent to lab for further quantitative analysis in order to represent the inflammation level in colon. Therefore, in our project, the fluorescence intensity will be the index that represents levels of gut inflammation.
Since then, we first needed to develop a standard quantitative measurement protocol, so we studied the following articles: Zong, Y et al (2017)[1] and Zhang, H. M. et al(2015).

<figure>

<img src="T--SHSBNU_China---gfp_zqd.png" width="800" height="100"/> <figcaption> Figure 1.We constructed our reporter circuit with sfgfp and pTac promoter </figcaption> </figure> <p>Following reference, we determined the experimental procedure:
Flat-bottom 96-well plates and sealing film were used throughout the study. Bacteria harboring parts/circuits of interest were inoculated from plates to LB medium and grown overnight (8−12 h, 1000 rpm, 37 °C, mB100-40 Thermo Shaker). Ten microliters of each overnight culture was sequentially diluted into 130 μL of fresh medium twice; the total dilution fold was 196. After growing the diluted cultures for ∼3 h, we diluted the exponentially growing cultures 700-fold using fresh medium; the dilution process was as follows: 10 μL of cell culture is added to 130 μL of M9 medium, which is followed by diluting 3 μL of this into 147 μL. Then, cultivation continued (1000 rpm, 37 °C, mB100-40); atspecifictimepoints,a2−50μL aliquot of each culture was transferred to a new plate containing 200 μL of PBS with 2 mg/mL kanamycin preadded to terminate protein expression. For the time course of cell growth after 700-fold dilution, OD 600 was recorded using Varioskan Flash (Thermal Scientific); the time interval was 5 min. <figure> <p style="text-align:center;"><img src="T--SHSBNU_China---characterization-step.jpeg" width="800" height="200"/> <figcaption> Figure 2.Experimental procedure for recording the time course of gene expression using flow cytometry. </figcaption> </figure> <p>The graph below shows our result.

<figure>

<img src="T--SHSBNU_China---Ptac-result.png" width = "900" height ="400"/> <figcaption> Figure 1.Ptac response curve in medium copy number vector. </figcaption> </figure> 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 170