Difference between revisions of "Part:BBa K3332079"
 
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<partinfo>BBa_K3332079 short</partinfo>
 
<partinfo>BBa_K3332079 short</partinfo>
  
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It can express EYFP without arabinose and express little EYFP induced by arabinose.
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It can express EYFP without arabinose and express little EYFP with arabinose.
  
 
===Usage and Biology===
 
===Usage and Biology===
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[[File:T-XMU-BBa K3332079.circuit.png|none|500px|caption]]
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<html>
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    <figure>
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This circuit was designed to test EYFP expression under inverter and inducible promoter: proBAD/araC. By comparing the fluorescence intensity of induced and non-induced to characterize the function of the inverter.
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        <img src="https://2020.igem.org/wiki/images/e/ed/T--XMU-China--XMU-China_2020-pBAD_inverter_eyfp_B0015.png" width="60%" style="float:center">
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        <figcaption>
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        <p style="font-size:1rem">
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        </p>
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        </figcaption>
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    </figure>
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</html>
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'''Fig 1'''. pBAD_inverter_eyfp_B0015(<partinfo>BBa_K3332079</partinfo>)
  
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This circuit was designed to test EYFP expression under inverter and inducible promoter: proBAD/araC. By comparing the fluorescence intensity/OD<sub>600</sub> of induction and non-induction bacteria, we managed to characterize the function of the inverter(<partinfo>BBa_Q04510</partinfo>).
  
 
===Characterization===
 
===Characterization===
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When we were building this circuit, we did the nucleic acid gel electrophoresis experiment to verify. After the circuit was built, we sent the plasmid to sequence, and got the correct sequencing. After new molecular cloning experiments, we did Enzyme-Cut identification to certify the plasmid is correct. We used the ''EcoR'' I and ''Pst'' I to cut the plasmid, then we got the target separate fragment.
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When we were building this circuit, we did the nucleic acid gel electrophoresis experiment to verify the ligation. After the circuit was built, we sent the plasmid to sequence, and got the correct result. After new molecular cloning experiments, we did Enzyme-Cut identification for further confirmation. We used the ''Eco''R I and ''Pst'' I to cut the plasmid, then we got the target separate fragment.
 
[[File:T-XMU-BBa K3332079.ger.png|none|500px|caption]]  
 
[[File:T-XMU-BBa K3332079.ger.png|none|500px|caption]]  
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Fig.2 pBAD_inverter_EYFP_pSB1C3 (BBa_K3332079) digested by ''EcoR'' I and ''Pst'' I
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'''Fig.2''' pBAD_inverter_EYFP_pSB1C3 (BBa_K3332079) digested by ''Eco''R I and ''Pst'' I (about 3000bp)
  
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Protocol:  
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'''Protocol: '''
  
 
1. Preparation of stock solution
 
1. Preparation of stock solution
  
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Dissolve arabinose in ddH2O to make 100× stock solution（the work concentration is 0.2%）
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Dissolve arabinose in ddH<sub>2</sub>O to make 100× stock solution（the work concentration is 0.2%）
  
 
2.Culture glycerol bacteria containing the corresponding plasmid in test tube for 12h.
 
2.Culture glycerol bacteria containing the corresponding plasmid in test tube for 12h.
  
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3.Add 4mL of the above bacterial solution into 200 mL LB medium and maintain the culture condition at 37 ℃ and 180 rpm.
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3.Add 4 mL of the above bacterial solution into 200 mL LB medium and maintain the culture condition at 37 ℃ and 180 rpm.
  
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4.Add 2 mL arabinose stock solution into the induction group when OD600 increased to 0.6.  
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4.Add 2 mL arabinose stock solution into the induction group when OD<sub>600</sub> increased to 0.6.  
  
 
5.Induce for 6 hours and the condition is the same as before.
 
5.Induce for 6 hours and the condition is the same as before.
  
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6.Then, sampling culture in 96-well plate reader to measure the fluorescence intensity (EYFP) and corresponding OD600, then calculate the fluorescence / OD value of each group.
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6.Then, sampling culture in 96-well plate reader to measure the fluorescence intensity (EYFP) and corresponding OD<sub>600</sub>, then calculate the fluorescence / OD value of each group.
  
 
Here is the result:
 
Here is the result:
  
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[[File:T--XMU-2082.fig.3.dem.png|none|500px|caption]]
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<html>
 +
    <figure>
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        <img src="https://2020.igem.org/wiki/images/3/3d/T--XMU-China--XMU-China_2020-BY_BNY_fluorescence.png" width="60%" style="float:center">
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        <figcaption>
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        <p style="font-size:1rem">
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        </p>
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        </figcaption>
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    </figure>
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</html>
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'''Fig 3.''' Fluorescence intensity/OD<sub>600</sub> for induction and non-induction group (6 hours). Data are collected and analyzed according to iGEM standard data analysis form after 6 hours of induction.
  
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We discovered that the downstream gene of proBAD/araC can be expressed in the presence of arabinose and decrease expression without arabinose. When the inverter is added to the circuit, the effect is reversed.
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We found that the downstream gene of proBAD/araC can be expressed when induced by arabinose, while the expression of EYFP decreases without the inducer. Besides, as we can see, the purple bar is higher on the right side(when the arabinose is absent). Therefore, we can come to the conclusion that when the inverter(<partinfo>BBa_Q04510</partinfo>) is added, the effect is reversed.
  
 
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Latest revision as of 22:08, 27 October 2020


pBAD/araC-Inverter-EYFP-terminator

It can express EYFP without arabinose and express little EYFP with arabinose.

Usage and Biology

Fig 1. pBAD_inverter_eyfp_B0015(BBa_K3332079)

This circuit was designed to test EYFP expression under inverter and inducible promoter: proBAD/araC. By comparing the fluorescence intensity/OD600 of induction and non-induction bacteria, we managed to characterize the function of the inverter(BBa_Q04510).

Characterization

When we were building this circuit, we did the nucleic acid gel electrophoresis experiment to verify the ligation. After the circuit was built, we sent the plasmid to sequence, and got the correct result. After new molecular cloning experiments, we did Enzyme-Cut identification for further confirmation. We used the EcoR I and Pst I to cut the plasmid, then we got the target separate fragment.

caption

Fig.2 pBAD_inverter_EYFP_pSB1C3 (BBa_K3332079) digested by EcoR I and Pst I (about 3000bp)

Protocol:

1. Preparation of stock solution

Dissolve arabinose in ddH2O to make 100× stock solution（the work concentration is 0.2%）

2.Culture glycerol bacteria containing the corresponding plasmid in test tube for 12h.

3.Add 4 mL of the above bacterial solution into 200 mL LB medium and maintain the culture condition at 37 ℃ and 180 rpm.

4.Add 2 mL arabinose stock solution into the induction group when OD600 increased to 0.6.

5.Induce for 6 hours and the condition is the same as before.

6.Then, sampling culture in 96-well plate reader to measure the fluorescence intensity (EYFP) and corresponding OD600, then calculate the fluorescence / OD value of each group.

Here is the result:

Fig 3. Fluorescence intensity/OD600 for induction and non-induction group (6 hours). Data are collected and analyzed according to iGEM standard data analysis form after 6 hours of induction.

We found that the downstream gene of proBAD/araC can be expressed when induced by arabinose, while the expression of EYFP decreases without the inducer. Besides, as we can see, the purple bar is higher on the right side(when the arabinose is absent). Therefore, we can come to the conclusion that when the inverter(BBa_Q04510) is added, the effect is reversed.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1205
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1144
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 979
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 961