Difference between revisions of "Part:BBa R0080"

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==Contribution: iGEM14_NUDT_CHINA==
 
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'''Group:''' iGEM14_NUDT_CHINA/ '''Author:''' Xinyuan Qiu
 
  
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'''Summary:''' We tested expression of this part in ''E.coli'', and corrected the founction description of this part.
 
  
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===Materials and Methods===
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==Functional Parameters: Austin_UTexas==
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We used the circuit of BBa_K1532008 to test the function of this part, the sequence and features of the part BBa_K1532008 is as follows:
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<html>
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https://static.igem.org/mediawiki/2014/f/f9/NUDT_CHINA_2014_K1532008.png
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<body>
 +
<partinfo>BBa_R0080 parameters</partinfo>
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<h3><center>Burden Imposed by this Part:</center></h3>
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<figure>
 +
<div class = "center">
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<center><img src = "https://static.igem.org/mediawiki/parts/f/fa/T--Austin_Utexas--no_burden_icon.png" style = "width:160px;height:120px"></center>
 +
</div>
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<figcaption><center><b>Burden Value: 0.5 ± 1.4% </b></center></figcaption>
 +
</figure>
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<p> Burden is the percent reduction in the growth rate of <i>E. coli</i> cells transformed with a plasmid containing this BioBrick (± values are 95% confidence limits). This BioBrick did not exhibit a burden that was significantly greater than zero (i.e., it appears to have little to no impact on growth). Therefore, users can depend on this part to remain stable for many bacterial cell divisions and in large culture volumes. Refer to any one of the
 +
<a href="https://parts.igem.org/Part:BBa_K3174002">BBa_K3174002</a> - <a href="https://parts.igem.org/Part:BBa_K3174007">BBa_K3174007</a> pages for more information on the methods, an explanation of the sources of burden,  and other conclusions from a large-scale measurement project conducted by the <a href="http://2019.igem.org/Team:Austin_UTexas">2019 Austin_UTexas team</a>.</p>
 +
<p>This functional parameter was added by the <a href="https://2020.igem.org/Team:Austin_UTexas/Contribution">2020 Austin_UTexas team.</a></p>
 +
</body>
 +
</html>
  
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Fluorometric measurement were processed with Fluoroskan(R) Ascent FL from ThermoTM using the filters of 485nm and 538nm. The E.coli to be tested was cultured in liquid LB media containing 35μg/mL chloramphenicol to OD1.6, than split into 96 well plates (from ThermoTM) by 150μL/well. The plate was then put into the Fluoroskan(R) Ascent FL and cultured in 25℃ and background shake of 90rpms. The Fluorometric measurement was processed with the interval time of 10 minutes.
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==2020 AHUT-ZJU-China’s Characterization==
  
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The layout of the plate is as follows:
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<!DOCTYPE html>
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<html lang="en">
  
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https://static.igem.org/mediawiki/2014/5/50/NUDT_CHINA_R0080_CON1.jpg
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<head>
 +
    <style>
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        #touniprot {
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            color: black;
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            text-decoration: underline;
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        }
  
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        #touniprot:hover {
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            color: blue;
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        }
  
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We draw the curve using the intensity figure of different amount of Arabinose subtracted by the figures of those without Arabinose.
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        #touniprot:visited {
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            color: purple;
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===Results===
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        }
  
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The result is as follows:
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        img {
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            max-width: 500px;
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            max-height: 500px;
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        }
  
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https://static.igem.org/mediawiki/2014/8/89/NUDT_CHINA_2014_RCC80_CON2.jpg
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        .fignote {
 +
            font-size: 12px;
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        }
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    </style>
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</head>
  
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The results indicates that with the existence of Arabinose and AraC, the part BBa_R0080 can activate the down-stream expression observably.
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<body>
  
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<!-- Uncomment this to enable Functional Parameter display
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    <p style="font-size: 16px;"><b>The Optimal Arabinose Concentration and Temperature for the Expression of Protein in <i>E.coli</i> BL21 (DE3) with the Promoter BBa_R0080 and pSB1C3 and the Relation
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===Functional Parameters===
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            between the Strength of BBa_R0080 and Arabinose Concentration at the Transcription Level.</b></p>
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<partinfo>BBa_R0080 parameters</partinfo>
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<!-- -->
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    <h3>Overview</h3>
 +
    <p>
 +
        In 2020, AHUT-ZJU-China constructed an EGFP expression vector (with pSB1C3 as the plasmid skeleton) in which the expression of EGFP was controlled by the promoter BBa_R0080 and transformed it
 +
        into <i>E.coli</i> BL21 (DE3) which is commonly used to express protein. Then, by measuring the EGFP fluorescence, we investigate the optimal arabinose concentration and temperature for the
 +
        expression of protein in <i>E.coli</i> BL21 (DE3) using the BBa_R0080 in pSB1C3.
 +
    </p>
 +
    <h3>Results</h3>
 +
 
 +
    <p>
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        Our part vectors contained EGFP were successfully constructed and sequence results were correct. Then we picked the positive clone for further experiment. When the <i>E.coli</i> BL21(DE3) was
 +
        cultured at the OD600 between 0.6 to 0.8, we added arabinose in different concentration and cultured the <i>E.coli</i> in different temperature. Absolute fluorescence and OD600 assessed by
 +
        fluorescence microplate reader was shown below.
 +
    </p>
 +
 
 +
    <div style="text-align: center;">
 +
        <img src="https://2020.igem.org/wiki/images/3/33/T--AHUT-ZJU-China--R0080_fig1a.png" alt="">
 +
    </div>
 +
 
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    <div style="text-align: center;">
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        <img src="https://2020.igem.org/wiki/images/d/d2/T--AHUT-ZJU-China--R0080_fig1b.png" alt="">
 +
    </div>
 +
 
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    <div style="text-align: center;">
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        <p class="fignote"><span style="font-weight: bold;color: black;">Fig1.</span>&nbsp;Fluorescence of EGFP under different treatment condition. Fig 1(A) (B) represents the treatment at 25℃, 30℃
 +
            respectively. The horizontal axis shows the different concentrations of Arabinose, the vertical axis shows the green fluorescence per OD600 (excitation wavelength: 485 nm; detection
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            wavelength: 528 nm), and segments and data points of different colors show the different culture temperature. Error bar indicates the standard error of replicates.</p>
 +
    </div>
 +
 
 +
    <br>
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    <p>From Fig1 we can learn that compare with 25℃, BBa_R0080 has as higher expression level under 30℃. And the optimum concentration under 30℃ is 0.1% of arabinose.</p>
 +
 
 +
    <h3>Protocol</h3>
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    <span style="font-weight: bolder;">Fluorescence Characterization</span>
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    <p>1. Linearize pSB1c3 plasmid by PCR.
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        (F: TACTAGTAGCGGCCGCTGCAG,R: CTCTAGAAGCGGCCGCGAATTC)
 +
        <br>
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        2. Obtain the sequence of BBa_R0080 from 2019 DNA Distribution Kit by PCR.
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        (F: GAATTCGCGGCCGCTTCTAGAG, R: CTGCAGCGGCCGCTACTAGTA)
 +
        <br>
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        3. Obtain the RBS+EGFP sequence and part sequence by PCR. Plasmid contained EGFP sequence was kindly donated by Associated Prof. Wentao Jin, sequence was identical to <a
 +
            href="https://www.uniprot.org/uniprot/A0A2V2QJP9" target="_blank" id="touniprot">UniProtKB - A0A2V2QJP9</a>
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        (F:AAAGAGGAGAAATACTAGATGAGCAAGGGC, R:TTACTTGTACAGCTCGTCCATG)
 +
        <br>
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        4. Use homologous recombination to construct the BBa_R0080-EGFP pasmid
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        <br>
 +
 
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        5. Transform the recombination plasmids into <span style="font-style: italic;">E.coli</span> DH5α, then pick the positive clones and sequence.
 +
        <br>
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        6. After recombinational sequence results were comfirmed, transform the plasmids into <span style="font-style: italic;">E.coli</span> BL21(DE3).
 +
        <br>
 +
        7. Coated the transformed BL21 onto the plate and added 1mM IPTG.
 +
        <br>
 +
        8. Incubated at 37℃ overnight and observed colony color under the blue light to see if the EGFP is expressed
 +
        <br>
 +
        9. Picked greener single colonies, add them into 1mL Lb, incubate at 37℃ in a shaker for 6-8h till the OD600 reach 0.6-0.8.
 +
        <br>
 +
        10. Add 50μl germ solution from last step into 5mL Lb, add IPTG in different concentration and induce for 5h at 25℃, 30℃.
 +
        <br>
 +
        11. Measure the fluorescence (excitation wavelength: 485 nm; detection wavelength: 528 nm) and OD600.
 +
    </p>
 +
</body>
 +
 
 +
</html>

Latest revision as of 13:08, 26 October 2020

Promoter (AraC regulated)

AraC operator, truncated to include araO1, araI1, araI2, c-amp1, and c-amp2 sites. This operator should *activate* transcription in the presence of AraC or the simple sugar [http://openwetware.org/wiki/Arabinose arabinose]

b/c the operator lacks the araO2 site, there should not be araC-mediated repression.


Usage and Biology

araC binding region from E.coli


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 103
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]



Functional Parameters: Austin_UTexas

BBa_R0080 parameters

Burden Imposed by this Part:

Burden Value: 0.5 ± 1.4%

Burden is the percent reduction in the growth rate of E. coli cells transformed with a plasmid containing this BioBrick (± values are 95% confidence limits). This BioBrick did not exhibit a burden that was significantly greater than zero (i.e., it appears to have little to no impact on growth). Therefore, users can depend on this part to remain stable for many bacterial cell divisions and in large culture volumes. Refer to any one of the BBa_K3174002 - BBa_K3174007 pages for more information on the methods, an explanation of the sources of burden, and other conclusions from a large-scale measurement project conducted by the 2019 Austin_UTexas team.

This functional parameter was added by the 2020 Austin_UTexas team.

2020 AHUT-ZJU-China’s Characterization

<!DOCTYPE html>

The Optimal Arabinose Concentration and Temperature for the Expression of Protein in E.coli BL21 (DE3) with the Promoter BBa_R0080 and pSB1C3 and the Relation between the Strength of BBa_R0080 and Arabinose Concentration at the Transcription Level.

Overview

In 2020, AHUT-ZJU-China constructed an EGFP expression vector (with pSB1C3 as the plasmid skeleton) in which the expression of EGFP was controlled by the promoter BBa_R0080 and transformed it into E.coli BL21 (DE3) which is commonly used to express protein. Then, by measuring the EGFP fluorescence, we investigate the optimal arabinose concentration and temperature for the expression of protein in E.coli BL21 (DE3) using the BBa_R0080 in pSB1C3.

Results

Our part vectors contained EGFP were successfully constructed and sequence results were correct. Then we picked the positive clone for further experiment. When the E.coli BL21(DE3) was cultured at the OD600 between 0.6 to 0.8, we added arabinose in different concentration and cultured the E.coli in different temperature. Absolute fluorescence and OD600 assessed by fluorescence microplate reader was shown below.

Fig1. Fluorescence of EGFP under different treatment condition. Fig 1(A) (B) represents the treatment at 25℃, 30℃ respectively. The horizontal axis shows the different concentrations of Arabinose, the vertical axis shows the green fluorescence per OD600 (excitation wavelength: 485 nm; detection wavelength: 528 nm), and segments and data points of different colors show the different culture temperature. Error bar indicates the standard error of replicates.


From Fig1 we can learn that compare with 25℃, BBa_R0080 has as higher expression level under 30℃. And the optimum concentration under 30℃ is 0.1% of arabinose.

Protocol

Fluorescence Characterization

1. Linearize pSB1c3 plasmid by PCR. (F: TACTAGTAGCGGCCGCTGCAG,R: CTCTAGAAGCGGCCGCGAATTC)
2. Obtain the sequence of BBa_R0080 from 2019 DNA Distribution Kit by PCR. (F: GAATTCGCGGCCGCTTCTAGAG, R: CTGCAGCGGCCGCTACTAGTA)
3. Obtain the RBS+EGFP sequence and part sequence by PCR. Plasmid contained EGFP sequence was kindly donated by Associated Prof. Wentao Jin, sequence was identical to UniProtKB - A0A2V2QJP9 (F:AAAGAGGAGAAATACTAGATGAGCAAGGGC, R:TTACTTGTACAGCTCGTCCATG)
4. Use homologous recombination to construct the BBa_R0080-EGFP pasmid
5. Transform the recombination plasmids into E.coli DH5α, then pick the positive clones and sequence.
6. After recombinational sequence results were comfirmed, transform the plasmids into E.coli BL21(DE3).
7. Coated the transformed BL21 onto the plate and added 1mM IPTG.
8. Incubated at 37℃ overnight and observed colony color under the blue light to see if the EGFP is expressed
9. Picked greener single colonies, add them into 1mL Lb, incubate at 37℃ in a shaker for 6-8h till the OD600 reach 0.6-0.8.
10. Add 50μl germ solution from last step into 5mL Lb, add IPTG in different concentration and induce for 5h at 25℃, 30℃.
11. Measure the fluorescence (excitation wavelength: 485 nm; detection wavelength: 528 nm) and OD600.