Difference between revisions of "Part:BBa K1402010"
 
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    <head>
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</head>
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        <style>
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            #touniprot {
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<body>
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                color: black;
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    <h1>2020 ZJU-China’s Characterization</h1>
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                text-decoration: underline;
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    <hr>
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            }
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    <p style="font-size: 20px;font-weight: bold;">The Optimal IPTG Concentration and Temperature for the Expression of Protein in<span style="font-style: italic;">E.coli</span>BL21 (DE3) with the
+
   
−
        Promoter BBa_K1402010 and pSB1C3 and the Relation between the Strength of BBa_R0011 and IPTG Concentration at the Transcription Level</p>
+
            #touniprot:hover {
−
    <br>
+
                color: blue;
−
    <h3>Overview</h3>
+
            }
−
    <p>In 2020, 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_K1402010 and transformed it
+
   
−
        into <span style="font-style: italic;">E.coli</span> BL21 (DE3) which is commonly used to express protein. Then, by measuring the EGFP fluorescence, we investigate the optimal IPTG
+
            #touniprot:visited {
−
        concentration and temperature for the expression of protein in <span style="font-style: italic;">E.coli</span> BL21 (DE3) using the BBa_K1402010 in pSB1C3. Meanwhile, we carried out qRT-PCR to
+
                color: purple;
−
        directly investigate the relation between the original strength of BBa_K1402010 and IPTG concentration at the transcription level without interfering by context.</p>
+
            }
−
    <br>
+
   
−
    <h3>Vector construction</h3>
+
            img {
−
    <p>Our part vectors contained EGFP were successfully constructed and sequence results were correct. With the induction of IPTG, as shown in Fig1, we can see the green fluorescence obviously under
+
                max-width: 500px;
−
        the stimulate of blue light.</p>
+
                max-height: 500px;
−
     <br>
+
            }
−
     <h3>Fluorescence result</h3>
+
   
−
    <p>Then we picked the positive clone in Fig 1 for further experiment. When the E.coli BL21(DE3) was cultured at the OD600 between 0.6 to 0.8, we added IPTG in different concentration and cultured
+
            .fignote {
−
        the E.coli in different temperature. Absolute fluorescence and OD600 assessed by fluorescence microplate reader was shown below.</p>
+
                font-size: 12px;
−
     <p>As shown in Fig 2 above, we can find that even through without induction of IPTG, fluorescence can be measured, suggested that when BBa_K1402010 was used to express protein in E.coli BL21 (DE3)
+
            }
−
        with pSB1C3 as the plasmid skeleton, it is leaky. But the leaky expression is unstable and it is slower than the degradation rate in some condition as shown in Fig 2(A) red line.</p>
+
        </style>
−
 
+
    </head>
−
     <p>Compared the expression level in different temperature, fluorescence in 37℃ was much higher than in 25℃ and 30℃, indicating that the optimum temperature for BBa_K1402010 expressed in BL21 with
+
   
−
        pSB1C3 as the skeleton is 37℃. And the optimum IPTG concentration in 37℃ was 2mM.</p>
+
    <body>
−
 
+
        <h1>2020 ZJU-China’s Characterization</h1>
−
     <br>
+
        <p style="font-size: 16px;font-weight: bold;">The Optimal IPTG Concentration and Temperature for the Expression of Protein in<span style="font-style: italic;">E.coli</span>BL21 (DE3) with the
−
    <h3>qRT-PCR result</h3>
+
            Promoter BBa_K1402010 and pSB1C3 and the Relation between the Strength of BBa_K1402010 and IPTG Concentration at the Transcription Level.</p>
−
    <p>From the results of fluorescence characterization, 37℃ seems to be a more favorable temperature for the opening of BBa_K1402010 in pSB1C3 in E.coli BL21 (DE3), so we chose this temperature as
+
        <br>
−
        the culture temperature for qRT-PCR. We selected a frequently used housekeeping genes 16S rRNA as reference genes to compare the relative expression level of EGFP controlled by BBa_ K1402010
+
        <h3>Overview</h3>
−
        under different IPTG concentrations of 1 mM, 1.5 mM, 2 mM, 2.5 mM and 3 mM at 37℃. </p>
+
        <p>In 2020, 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_K1402010 and transformed it
−
 
+
            into <span style="font-style: italic;">E.coli</span> BL21 (DE3) which is commonly used to express protein. Then, by measuring the EGFP fluorescence, we investigate the optimal IPTG
−
     <p>From Fig 3, we can conclude that the optimum IPTG concentration is 2.5mM, which is corresponding to the result in Fig 2(C). However, the expression level between 1mM to 2mM IPTG showed a slow
+
            concentration and temperature for the expression of protein in <span style="font-style: italic;">E.coli</span> BL21 (DE3) using the BBa_K1402010 in pSB1C3. Meanwhile, we carried out RT-qPCR to
−
        downward trend. We speculated that the actual expression level from 1mM to 2mM IPTG were very closed due to both of them were not the most suitable for BBa_K1402010, but the degradation rate
+
            directly investigate the relation between the original strength of BBa_K1402010 and IPTG concentration at the transcription level without interfering by context.</p>
−
        caused by different bacterial denisity were different, resulted in a slow downward trend.</p>
+
        <br>
−
 
+
        <h3>Vector construction</h3>
−
     <br>
+
        <p>Our part vectors contained EGFP were successfully constructed and sequence results were correct. With the induction of IPTG, as shown in Fig1, we can see the green fluorescence obviously under
−
    <h3>Protocol</h3>
+
            the stimulate of blue light.</p>
−
    <span style="font-weight: bolder;">Fluorescence Characterization</span>
+
      
−
    <p>1. Linearize pSB1c3 plasmid by PCR.
+
        <div style="text-align: center;">
−
        (F: TACTAGTAGCGGCCGCTGCAG,R: CTCTAGAAGCGGCCGCGAATTC)
+
            <img src="https://2020.igem.org/wiki/images/0/0a/T--ZJU-China--wiki_measure_fig1.jpg" alt="">
−
        2. Obtain the sequence of BBa_K1402010 from 2019 DNA Distribution Kit by PCR.
+
        </div>
−
        (F: GAATTCGCGGCCGCTTCTAGAG, R: CTGCAGCGGCCGCTACTAGTA)
+
        <div style="text-align: center;">
−
        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
+
            <p class="fignote"><span style="font-weight: bold;color: black;">Fig1.</span>&nbsp;Fluorescence of EGFP after the promoter BBa_K1402010 with the induction of IPTG.</p>
−
        (链接到：https://www.uniprot.org/uniprot/A0A2V2QJP9)
+
        </div>
−
        (F:AAAGAGGAGAAATACTAGATGAGCAAGGGC, R:TTACTTGTACAGCTCGTCCATG)
+
      
−
        4. Use homologous recombination to construct the BBa_K1402010-EGFP pasmid
+
        <br>
−
        5. Transform the recombination plasmids into E.coli DH5α, then pick the positive clones and sequence.
+
        <h3>Fluorescence result</h3>
−
        6. After recombinational sequence results were comfirmed, transform the plasmids into E.coli BL21(DE3).
+
        <p>Then we picked the positive clone in Fig 1 for further experiment. When the <span style="font-style: italic;">E.coli</span> BL21(DE3) was cultured at the OD600 between 0.6 to 0.8, we added IPTG
−
        7. Coated the transformed BL21 onto the plate and added 1mM IPTG.
+
            in different concentration and cultured
−
        8. Incubated at 37℃ overnight and observed colony color under the blue light to see if the EGFP is expressed
+
            the <span style="font-style: italic;">E.coli</span> in different temperature. Absolute fluorescence and
−
        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.
+
            OD600 assessed by fluorescence microplate reader was shown below.</p>
−
        10. Add 50μl germ solution from last step into 5mL Lb, add IPTG in different concentration and induce for 5h at 25℃, 30℃ and 37℃.
+
        <div style="text-align: center;">
−
        11. Measure the fluorescence (excitation wavelength: 485 nm; detection wavelength: 528 nm) and OD600.
+
            <img src="https://2020.igem.org/wiki/images/a/ab/T--ZJU-China--wiki_measure_fig2a.jpg" alt="">
−
    </p>
+
            <img src="https://2020.igem.org/wiki/images/5/5f/T--ZJU-China--wiki_measure_fig2b.jpg" alt="">
−
    <span>qRT-PCR</span>
+
            <img src="https://2020.igem.org/wiki/images/8/8e/T--ZJU-China--wiki_measure_fig2c.jpg" alt="">
−
    <p>
+
        </div>
−
        1. Pick a single colony from BBa_ K1402010 and inoculate in 5mL liquid LB medium + 5μL Chloramphenicol (25mg/mL in EtOH) and grow the cells overnight at 37 °C and 220 rpm.
+
        <p class="fignote"><span style="font-weight: bolder;color: black;">Fig2.&nbsp;Fluorescence of EGFP under different treatment condition.</span> Fig 2(A) (B) (C) represents the treatment at 25℃, 30℃
−
        2. Inoculate 50μL of the overnight culture into 4.95 mL LB with Chloramphenicol to make 7 parallel groups and grow the cells at 37 °C and 200 rpm until OD600 reaches 0.6.
+
            and 37℃ respectively. The
−
        3. Add IPTG to final concentrations of 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3mM IPTG respectively and grow the cells at temperature of 37℃ and 200 rpm for 5 hours.
+
            horizontal axis shows the different concentrations of IPTG, the vertical axis shows the green fluorescence per OD600 (excitation wavelength: 485 nm; detection wavelength: 528 nm), and segments
−
        4. Extracted RNA, make reverse transcription and carry out qRT-PCR with 16S rRNA as reference genes. qRT-PCR data was proceeded by Bio-Rad CFX Maestro.
+
            and
−
        qRT-PCR primer for 16srRNA:
+
            data points of different colors show the different culture temperature. Error bar indicates the standard error of replicates.</p>
−
 
+
      
−
        qRT-PCR primer for EGFP:
+
        <p>As shown in Fig 2 above, we can find that even through without induction of IPTG, fluorescence can be measured, suggested that when BBa_K1402010 was used to express protein in <span
−
        F: AGATCCGCCACAACATCGAG, R: AACTCCAGCAGGACCATGTG
+
                style="font-style: italic;">E.coli</span> BL21 (DE3)
−
        qRT-PCR primer for 16srRNA:
+
            with pSB1C3 as the plasmid skeleton, it is leaky. But the leaky expression is unstable and it is slower than the degradation rate in some condition as shown in Fig 2(A) red line.</p>
−
        F: AACACATGCAAGTCGAACGG, R: TAAGGTCCCCCTCTTTGTGC
+
      
−
 
+
        <p>Compared the expression level in different temperature, fluorescence in 37℃ was much higher than in 25℃ and 30℃, indicating that the optimum temperature for BBa_K1402010 expressed in BL21 with
−
    </p>
+
            pSB1C3 as the skeleton is 37℃. And the optimum IPTG concentration in 37℃ was 2mM.</p>
−
 
+
      
−
</body>
+
        <br>
−
 
+
        <h3>RT-qPCR result</h3>
−
</html>
+
        <p>From the results of fluorescence characterization, 37℃ seems to be a more favorable temperature for the opening of BBa_K1402010 in pSB1C3 in <span style="font-style: italic;">E.coli</span> BL21
 +
            (DE3), so we chose this temperature as the culture temperature for RT-qPCR. We selected a frequently used housekeeping genes 16S rRNA as reference genes to compare the relative expression
 +
            level of EGFP controlled by BBa_ K1402010
 +
            under different IPTG concentrations of 1 mM, 1.5 mM, 2 mM, 2.5 mM and 3 mM at 37℃. </p>
 +
      
 +
        <div style="text-align: center;">
 +
            <img src="https://2020.igem.org/wiki/images/7/7a/T--ZJU-China--wiki_measure_fig3.jpg" alt="">
 +
        </div>
 +
        <p class="fitnote"><span style="font-weight: bolder;color: black;">Fig3.&nbsp;The relative normalized expression level of EGFP of <i>E.coli</i> BL21 (DE3) under the conditions of IPTG concentration of 1
 +
                mM, 1.5mM, 2 mM, 2.5mM and 3
 +
                mM at 37℃. </span>The horizontal axis shows the different concentrations of IPTG, the vertical axis shows the relative normalized expression level of EGFP of each test group.</p>
 +
        <p>From Fig 3, we can conclude that the optimum IPTG concentration is 2.5mM, which is corresponding to the result in Fig 2(C). However, the expression level between 1mM to 2mM IPTG showed a slow
 +
            downward trend. We speculated that the actual expression level from 1mM to 2mM IPTG were very closed due to both of them were not the most suitable for BBa_K1402010, but the degradation rate
 +
            caused by different bacterial denisity were different, resulted in a slow downward trend.</p>
 +
      
 +
        <br>
 +
        <h3>Protocol</h3>
 +
        <span style="font-weight: bolder;">Fluorescence Characterization</span>
 +
        <p>1. Linearize pSB1c3 plasmid by PCR.
 +
            (F: TACTAGTAGCGGCCGCTGCAG,R: CTCTAGAAGCGGCCGCGAATTC)
 +
            <br>
 +
            2. Obtain the sequence of BBa_K1402010 from 2019 DNA Distribution Kit by PCR.
 +
            (F: GAATTCGCGGCCGCTTCTAGAG, R: CTGCAGCGGCCGCTACTAGTA)
 +
            <br>
 +
            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>
 +
            (F:AAAGAGGAGAAATACTAGATGAGCAAGGGC, R:TTACTTGTACAGCTCGTCCATG)
 +
            <br>
 +
            4. Use homologous recombination to construct the BBa_K1402010-EGFP pasmid
 +
            <br>
 +
   
 +
            5. Transform the recombination plasmids into <span style="font-style: italic;">E.coli</span> DH5α, then pick the positive clones and sequence.
 +
            <br>
 +
            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℃ and 37℃.
 +
            <br>
 +
            11. Measure the fluorescence (excitation wavelength: 485 nm; detection wavelength: 528 nm) and OD600.
 +
        </p>
 +
        <span style="font-weight: bolder;">RT-qPCR</span>
 +
        <p>
 +
            1. Pick a single colony from BBa_ K1402010 and inoculate in 5mL liquid LB medium + 5μL Chloramphenicol (25mg/mL in EtOH) and grow the cells overnight at 37 °C and 220 rpm.
 +
            <br>
 +
            2. Inoculate 50μL of the overnight culture into 4.95 mL LB with Chloramphenicol to make 7 parallel groups and grow the cells at 37 °C and 200 rpm until OD600 reaches 0.6.
 +
            <br>
 +
            3. Add IPTG to final concentrations of 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3mM IPTG respectively and grow the cells at temperature of 37℃ and 200 rpm for 5 hours.
 +
            <br>
 +
            4. Extracted RNA, make reverse transcription and carry out RT-qPCR with 16S rRNA as reference genes. RT-qPCR data was proceeded by Bio-Rad CFX Maestro.
 +
            <br>
 +
            <br>
 +
            RT-qPCR primer for EGFP:
 +
            <br>
 +
            F: AGATCCGCCACAACATCGAG, R: AACTCCAGCAGGACCATGTG
 +
            <br>
 +
            <br>
 +
            RT-qPCR primer for 16srRNA:
 +
            <br>
 +
            F: AACACATGCAAGTCGAACGG, R: TAAGGTCCCCCTCTTTGTGC
 +
        </p>
 +
   
 +
    </body>
 +
   
 +
    </html>

Latest revision as of 13:50, 26 October 2020

Lac Promoter

This part is an inverting regulator sensitive to LacI (BBa_C0010, BBa_C0012, etc.) The T7-lac promoter is a hybrid regulatory region consisting of the T7 promoter of T7 phage next to LacO. The hybrid design allows for strong promotion that can nevertheless be: (1)repressed by LacI. (2)induced by lac or IPTG, showing a high expression level. (3)CAP protein will not influence the expression. It is a strong promoter. You can only use this part in E.Coli BL21(DE3).

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]


2020 ZJU-China’s Characterization

The Optimal IPTG Concentration and Temperature for the Expression of Protein inE.coliBL21 (DE3) with the Promoter BBa_K1402010 and pSB1C3 and the Relation between the Strength of BBa_K1402010 and IPTG Concentration at the Transcription Level.


Overview

In 2020, 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_K1402010 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 IPTG concentration and temperature for the expression of protein in E.coli BL21 (DE3) using the BBa_K1402010 in pSB1C3. Meanwhile, we carried out RT-qPCR to directly investigate the relation between the original strength of BBa_K1402010 and IPTG concentration at the transcription level without interfering by context.


Vector construction

Our part vectors contained EGFP were successfully constructed and sequence results were correct. With the induction of IPTG, as shown in Fig1, we can see the green fluorescence obviously under the stimulate of blue light.

Fig1. Fluorescence of EGFP after the promoter BBa_K1402010 with the induction of IPTG.


Fluorescence result

Then we picked the positive clone in Fig 1 for further experiment. When the E.coli BL21(DE3) was cultured at the OD600 between 0.6 to 0.8, we added IPTG in different concentration and cultured the E.coli in different temperature. Absolute fluorescence and OD600 assessed by fluorescence microplate reader was shown below.

Fig2. Fluorescence of EGFP under different treatment condition. Fig 2(A) (B) (C) represents the treatment at 25℃, 30℃ and 37℃ respectively. The horizontal axis shows the different concentrations of IPTG, 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.

As shown in Fig 2 above, we can find that even through without induction of IPTG, fluorescence can be measured, suggested that when BBa_K1402010 was used to express protein in E.coli BL21 (DE3) with pSB1C3 as the plasmid skeleton, it is leaky. But the leaky expression is unstable and it is slower than the degradation rate in some condition as shown in Fig 2(A) red line.

Compared the expression level in different temperature, fluorescence in 37℃ was much higher than in 25℃ and 30℃, indicating that the optimum temperature for BBa_K1402010 expressed in BL21 with pSB1C3 as the skeleton is 37℃. And the optimum IPTG concentration in 37℃ was 2mM.


RT-qPCR result

From the results of fluorescence characterization, 37℃ seems to be a more favorable temperature for the opening of BBa_K1402010 in pSB1C3 in E.coli BL21 (DE3), so we chose this temperature as the culture temperature for RT-qPCR. We selected a frequently used housekeeping genes 16S rRNA as reference genes to compare the relative expression level of EGFP controlled by BBa_ K1402010 under different IPTG concentrations of 1 mM, 1.5 mM, 2 mM, 2.5 mM and 3 mM at 37℃.

Fig3. The relative normalized expression level of EGFP of E.coli BL21 (DE3) under the conditions of IPTG concentration of 1 mM, 1.5mM, 2 mM, 2.5mM and 3 mM at 37℃. The horizontal axis shows the different concentrations of IPTG, the vertical axis shows the relative normalized expression level of EGFP of each test group.

From Fig 3, we can conclude that the optimum IPTG concentration is 2.5mM, which is corresponding to the result in Fig 2(C). However, the expression level between 1mM to 2mM IPTG showed a slow downward trend. We speculated that the actual expression level from 1mM to 2mM IPTG were very closed due to both of them were not the most suitable for BBa_K1402010, but the degradation rate caused by different bacterial denisity were different, resulted in a slow downward trend.


Protocol

Fluorescence Characterization

1. Linearize pSB1c3 plasmid by PCR. (F: TACTAGTAGCGGCCGCTGCAG,R: CTCTAGAAGCGGCCGCGAATTC)
2. Obtain the sequence of BBa_K1402010 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_K1402010-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℃ and 37℃.
11. Measure the fluorescence (excitation wavelength: 485 nm; detection wavelength: 528 nm) and OD600.

RT-qPCR

1. Pick a single colony from BBa_ K1402010 and inoculate in 5mL liquid LB medium + 5μL Chloramphenicol (25mg/mL in EtOH) and grow the cells overnight at 37 °C and 220 rpm.
2. Inoculate 50μL of the overnight culture into 4.95 mL LB with Chloramphenicol to make 7 parallel groups and grow the cells at 37 °C and 200 rpm until OD600 reaches 0.6.
3. Add IPTG to final concentrations of 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3mM IPTG respectively and grow the cells at temperature of 37℃ and 200 rpm for 5 hours.
4. Extracted RNA, make reverse transcription and carry out RT-qPCR with 16S rRNA as reference genes. RT-qPCR data was proceeded by Bio-Rad CFX Maestro.

RT-qPCR primer for EGFP:
F: AGATCCGCCACAACATCGAG, R: AACTCCAGCAGGACCATGTG

RT-qPCR primer for 16srRNA:
F: AACACATGCAAGTCGAACGG, R: TAAGGTCCCCCTCTTTGTGC