Difference between revisions of "Part:BBa K3332082"
 
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<partinfo>BBa_K3332082 short</partinfo>
 
<partinfo>BBa_K3332082 short</partinfo>
  
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It can express cyan fluorescent protein under certain conditions.It is used to characterize the function of the MazF as a control group
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It can express cyan fluorescent protein under certain conditions.It is used to characterize the function of the mazF and inverter as a control group
  
  
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     </figure>
 
     </figure>
 
</html>
 
</html>
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'''Fig 1.''' pBAD_B0034_eyfp_B0015
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'''Fig 1.''' pBAD_B0034_eyfp_B0015(<partinfo>BBa_K3332082</partinfo>)
  
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This composite part is used as a control group to demonstrate the effect of inverter, comparing with proBAD/araC-inverter-EYFP-terminator (BBa_K3332079). EYFP serves as a reporter gene. By comparing the fluorescence intensity/OD600 of induced and non-induced bacteria, we can characterize the function of the inverter.
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This composite part is used as a control group to demonstrate the effect of inverter(<partinfo>BBa_Q04510</partinfo>), comparing with proBAD/araC_inverter_EYFP_terminator (<partinfo>BBa_K3332079</partinfo>). EYFP serves as a reporter gene. By comparing the fluorescence intensity/OD<sub>600</sub> of induced and non-induced bacteria, we can 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 Spe 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 ''Spe'' I and ''Pst'' I to cut the plasmid, then we got the target separate fragment.
 
[[File:T--XMU-2082.fig.2.ger.png|none|500px|caption]]  
 
[[File:T--XMU-2082.fig.2.ger.png|none|500px|caption]]  
  
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Fig.2 proBAD/araC-B0034-E0030-B0015_pSB1C3 (BBa_K3332082) digested by ''Spe'' I and ''Pst'' I (about 4148 bp)
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'''Fig.2''' proBAD/araC_B0034_E0030_B0015_pSB1C3 (<partinfo>BBa_K3332082</partinfo>) digested by ''Spe'' I and ''Pst'' I (about 4148 bp)
  
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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%）
  
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2.Culture glycerol bacteria containing the corresponding plasmid in test tube for 12h.
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2.Culture glycerol bacteria containing the corresponding plasmid in test tube for 12 h.
  
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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>
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    <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.
  
  

Latest revision as of 22:05, 27 October 2020


pBAD/araC promoter-RBS-EYFP-terminator

It can express cyan fluorescent protein under certain conditions.It is used to characterize the function of the mazF and inverter as a control group


Usage and Biology

Fig 1. pBAD_B0034_eyfp_B0015(BBa_K3332082)

This composite part is used as a control group to demonstrate the effect of inverter(BBa_Q04510), comparing with proBAD/araC_inverter_EYFP_terminator (BBa_K3332079). EYFP serves as a reporter gene. By comparing the fluorescence intensity/OD600 of induced and non-induced bacteria, we can 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 Spe I and Pst I to cut the plasmid, then we got the target separate fragment.

caption

Fig.2 proBAD/araC_B0034_E0030_B0015_pSB1C3 (BBa_K3332082) digested by Spe I and Pst I (about 4148 bp)

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 12 h.

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