Difference between revisions of "Part:BBa K2916042"
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<partinfo>BBa_K2916042 short</partinfo>
 
<partinfo>BBa_K2916042 short</partinfo>
  
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This part is used for expression of RF3 needed for the PURE and OnePot PURE cell-free systems.
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This part is used for expression of Release Factor 3 needed for the OnePot PURE cell-free system.
  
 
<!-- Add more about the biology of this part here-->
 
<!-- Add more about the biology of this part here-->
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Used in OnePot PURE  
 
Used in OnePot PURE  
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===Characterization===
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=='''Expression and purification of RF3'''==
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<br/>
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RF3 is one of the proteins we used for the OnePot PURE cell-free system. We expressed it in M15 E.coli strain using a pQE30 vector. The expression system has a T5 lac operator, RBS and a lambda t0 Terminator, enabling us to regulate the expression with IPTG.<br/><br/>
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'''Methods'''
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<br/>
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 +
RF3 was purified using our <html><a style="padding: 0px; margin: 0px;" href="https://www.protocols.io/view/protein-purification-for-onepot-pure-cell-free-sys-8auhsew"> protocol </a></html>. To test if the protein was actually expressed, we performed a SDS-PAGE that is presented below. On the left side we can see the results included in the initial OnePot PURE paper (<i>Lavickova et al, 2019</i>) while on the right (batch1_a,b and batch2_a,b) are the solutions we produced ourselves. (The procedure we followed and the conditions of the experiment can be found <html><a style="padding: 0px; margin: 0px;" href="https://www.protocols.io/view/sps-page-protein-electrophoresis-775hrq6"> here</a></html>).
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<br/>
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<html>
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<figure style="text-align:center;">
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                <img style="max-width:700px;" src="https://2019.igem.org/wiki/images/b/bb/T--EPFL--ProteinMolec.png" alt="control">
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                <figcaption><b>Figure 1:</b> SDS-PAGE of OnePot PURE protein solution.</figcaption>
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              </figure>
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</html>
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'''Conclusion'''
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<br/> RF3 has a molecular weight of around 60kDa, but even though we cannot be absolutely sure if the band shown is only due to it, we may assume that it is expressed. To verify the existence and functionality of this protein we need to proceed with more experiments that would be mainly focused on the efficiency of the system.
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<br/>
 +
<br/>
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=='''OnePot PURE functionality test'''==
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 +
 +
<br/>
 +
To make sure that we have all the proteins in our OnePot PURE protein solution, and that they all function properly we need check if proteins can be expressed in our OnePot PURE cell-free system. <br/>
 +
 +
'''Methods'''<br/>
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 +
We expressed <html><a style="padding: 0px; margin: 0px;" href="https://parts.igem.org/Part:BBa_I746909"> superfolding GFP</a></html> following the <html><a style="padding: 0px; margin: 0px;" href="https://www.protocols.io/view/protein-expression-in-onepot-pure-cell-free-system-8avhse6"> protocol</a></html> we designed in 10μl reactions, and measured the fluorescence on a plate reader at excitation wavelength of 535nm. We tested the expression using different concentrations of the sf GFP DNA template and also compared it with the fluorescence produced in PURExpress from NEB.
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<br/>
 +
 +
 +
<html>
 +
<figure style="text-align:center;">
 +
                <img style="max-width:700px;" src="https://2019.igem.org/wiki/images/b/b2/T--EPFL--resultsOnePot10.png" alt="control">
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                <figcaption><b>Figure 2:</b> sf GFP expression using 10nM DNA template.</figcaption>
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              </figure>
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</html>
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<html>
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<figure style="text-align:center;">
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                <img style="max-width:700px;" src="https://2019.igem.org/wiki/images/7/75/T--EPFL--resultsOnePot5.png" alt="control">
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                <figcaption><b>Figure 3:</b> sf GFP expression using 5nM DNA template.</figcaption>
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              </figure>
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</html>
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<html>
 +
<figure style="text-align:center;">
 +
                <img style="max-width:700px;" src="https://2019.igem.org/wiki/images/e/e7/T--EPFL--resultsOnePot25.png" alt="control">
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                <figcaption><b>Figure 4:</b> sf GFP expression using 2.5nM DNA template.</figcaption>
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              </figure>
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</html>
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<html>
 +
<figure style="text-align:center;">
 +
                <img style="max-width:700px;" src="https://2019.igem.org/wiki/images/7/70/T--EPFL--resultsOnePot.png" alt="control">
 +
                <figcaption><b>Figure 5:</b> Comparison between OnePot PURE and PURExpress at saturation.</figcaption>
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              </figure>
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</html>
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'''Conclusion'''
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<br/> The expression was successful so we can confirm that RF3 exists in our protein solution and is also functioning properly.
 +
<br/>
 +
  
  

Revision as of 19:42, 21 October 2019


Expression of RF3 in E.coli

This part is used for expression of Release Factor 3 needed for the OnePot PURE cell-free system.


Usage and Biology

Used in OnePot PURE


Characterization

Expression and purification of RF3


RF3 is one of the proteins we used for the OnePot PURE cell-free system. We expressed it in M15 E.coli strain using a pQE30 vector. The expression system has a T5 lac operator, RBS and a lambda t0 Terminator, enabling us to regulate the expression with IPTG.

Methods

RF3 was purified using our protocol . To test if the protein was actually expressed, we performed a SDS-PAGE that is presented below. On the left side we can see the results included in the initial OnePot PURE paper (Lavickova et al, 2019) while on the right (batch1_a,b and batch2_a,b) are the solutions we produced ourselves. (The procedure we followed and the conditions of the experiment can be found here).

control
Figure 1: SDS-PAGE of OnePot PURE protein solution.

Conclusion
RF3 has a molecular weight of around 60kDa, but even though we cannot be absolutely sure if the band shown is only due to it, we may assume that it is expressed. To verify the existence and functionality of this protein we need to proceed with more experiments that would be mainly focused on the efficiency of the system.

OnePot PURE functionality test


To make sure that we have all the proteins in our OnePot PURE protein solution, and that they all function properly we need check if proteins can be expressed in our OnePot PURE cell-free system.

Methods

We expressed superfolding GFP following the protocol we designed in 10μl reactions, and measured the fluorescence on a plate reader at excitation wavelength of 535nm. We tested the expression using different concentrations of the sf GFP DNA template and also compared it with the fluorescence produced in PURExpress from NEB.


control
Figure 2: sf GFP expression using 10nM DNA template.
control
Figure 3: sf GFP expression using 5nM DNA template.
control
Figure 4: sf GFP expression using 2.5nM DNA template.
control
Figure 5: Comparison between OnePot PURE and PURExpress at saturation.

Conclusion
The expression was successful so we can confirm that RF3 exists in our protein solution and is also functioning properly.



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
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 979
    Illegal AgeI site found at 1040
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1203