Difference between revisions of "Part:BBa K2443063"
 
(One intermediate revision by the same user not shown)
Line 5: Line 5:
 
Initiation factor 1, has been Codon optimized for use in <i>Escherichia coli</i>. Contains a N-terminal histidine tag.  
 
Initiation factor 1, has been Codon optimized for use in <i>Escherichia coli</i>. Contains a N-terminal histidine tag.  
  
 +
=='''Expression of the IF1  - EPFL,2019'''==
  
 +
 +
<br/>
 +
''' Group: ''' <html><a style="padding: 0px; margin: 0px;" href="https://2019.igem.org/Team:EPFL"> EPFL, 2019</a></html>
 +
<br/>
 +
<br/>
 +
 +
'''Methods'''
 +
<br/>
 +
IF1 is one of the proteins we used for 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/>
 +
 +
IF1 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 expresses we performed a SDS-PAGE that is presented bellow. On the left side we can see the results included in the initial OnePot PURE paper (Lavickova,2019) while on the right (batch1_a,b and batch2_a,b) are the solutions we produced ourselves.
 +
<br/>
 +
 +
<html>
 +
<figure style="text-align:center;">
 +
                <img style="max-width:700px;" src="https://2019.igem.org/wiki/images/b/bb/T--EPFL--ProteinMolec.png" alt="control">
 +
                <figcaption><b>Figure 1:</b> SDS-PAGE of OnePot PURE protein solution.</figcaption>
 +
              </figure>
 +
</html>
 +
 +
'''Conclusion'''
 +
<br/> IF1 has a molecular weight of around 9kDa and since no other protein we purified has similar molecular weight, meaning the band in this region is solely produced due to IF1, we can confirm that the protein was expressed successfully using our system.
 +
<br/>
 +
 +
 +
<br/>
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===

Latest revision as of 18:36, 21 October 2019


IF1

Initiation factor 1, has been Codon optimized for use in Escherichia coli. Contains a N-terminal histidine tag.

Expression of the IF1 - EPFL,2019


Group: EPFL, 2019

Methods
IF1 is one of the proteins we used for 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.

IF1 was purified using our protocol . To test if the protein was actually expresses we performed a SDS-PAGE that is presented bellow. On the left side we can see the results included in the initial OnePot PURE paper (Lavickova,2019) while on the right (batch1_a,b and batch2_a,b) are the solutions we produced ourselves.

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

Conclusion
IF1 has a molecular weight of around 9kDa and since no other protein we purified has similar molecular weight, meaning the band in this region is solely produced due to IF1, we can confirm that the protein was expressed successfully using our system.



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]