Team:METU Turkey/Results Discussion/CooA Expression and Purification

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                       <a href="https://2010.igem.org/Team:METU_Turkey/Conclusion">Conclusion</a> |
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<p style="font-size:110%; color:#576f91; font-family:georgia,serif;"><br>  
<p style="font-size:110%; color:#576f91; font-family:georgia,serif;"><br>  
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<br> <h2>1-Overexpression optimization</h2>
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<br>1-Overexpression optimization
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<br> <h3>A. Expression vector comparison (pKK versus pTriEx)</h3>
 +
<br>When we compare the below results, the expression of pTriEx vector is higher than the expression of pTriEx vector
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<br>A. Expression vector comparison (pKK versus pTriEx)
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<br> <h3>A.1. Expression in BL21 with pTriEx</h3>
 +
<br> In this expression experiment, we obtained 2 different bands in BL21 pTriEx from BL21 negative control as indicated gel photo. CooA is found in two form (monomer and dimer).
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<br>When we compare the below results, the expression of pTriEx vector is higher than the expression of pTriEx vector.  
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<div align="center" style="margin:15px 0px 0px 0px"><img style="border: 0px solid ; width: 400px; height: 400px;" alt="w7" src="https://static.igem.org/mediawiki/2010/3/35/Br1.jpg"></a></div>
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<br>10/09/18
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<br> <h3>A.2. Expression in JM109 and BL21 with pKK vector</h3>
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<br>Expression in BL21 with pTriEx
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We failed to express pKK vector in JM109. We have a little expression in BL21.
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<br>In this expression experiment, we obtained 2 different bands in BL21 pTriEx from BL21 negative control as indicated gel photo. CooA is found in two form (monomer and dimer).
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<div align="center" style="margin:15px 0px 0px 0px"><img style="border: 0px solid ; width: 400px; height: 400px;" alt="w7" src="https://static.igem.org/mediawiki/2010/b/b9/Br2.jpg"></a></div>
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<br>10/10/05
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<br> <html><div><a href="https://2010.igem.org/Team:METU_Turkey/Results_Discussion/pKK_versus_pTriEx"><strong>Details...</strong></a></div></html>
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<br>Expression in JM109 and BL21 with pKK vector
 
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<br>In this expriment, for the first time we want to try CooA pKK vector in BL21. The vector was sent by Aono and he advised us to express it in JM109. But we have failed to express it. So we tried it in BL21. We have no result with JM109 and a little expression in BL21. Therefore; we gave up from expressing pKK vector in BL21. And JM109 grows slowly,also we never see any band in JM109 crude lysate in many unpublished data. Therefore; we lysated all bacteria with same OD600. But result wasn't changed.
 
 +
<br> <h3>B. Expression host comparison (JM109 versus BL21)</h3>
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<br>B. Expression host comparison (JM109 versus BL21)
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<br> BL21 is better host than JM109 for CooA.
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<br>When we look at the experiment 10/10/5, there is no expression with pKK in JM109 and we have greyed bands belongs to CooA as indicated gel photo of 10/10/5 experiment.
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<br> <h3>C. IPTG induction studies</h3>
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<br> Expression comparison with different IPTG Concentration of pTriEx vector.
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<html>
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<div align="center" style="margin:15px 0px 0px 0px"><img style="border: 0px solid ; width: 400px; height: 400px;" alt="w7" src="https://static.igem.org/mediawiki/2010/6/6a/Br3.jpg"></a></div></html>
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<br> Our CooA pTriEx vector is expressed without IPTG induction but is less than others. When 2mM IPTG was added to culture, expression of CooA was decreased. It has probably a toxic effect on our bacteria.
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<br>C. IPTG induction studies
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<br> <html><div><a href="https://2010.igem.org/Team:METU_Turkey/Results_Discussion/IPTG_induction"><strong>Details...</strong></a></div></html>
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<br>Expression comparison with different IPTG Concentration of pTriEx vector
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<br> <h3>D. Ferric Citrate effect</h3>
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<br>10/10/10
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<br> Time Dependent Expression of pTriEx vector  with Ferric Citrate and without Ferric Citrate
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<br>In expression comparison with IPTG concentration experiment, we realized that our CooA pTriEx vector is expressed without IPTG induction however when the cultures were induced with IPTG the expression incresed a little. Thus, we concluded that IPTG concentration doesn't much affect our expression yield. However, when 2mM IPTG was added to culture,expression of CooA was decreased. It has probably a toxic affect on our bacteria.
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<html>
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<div align="center" style="margin:15px 0px 0px 0px"><img style="border: 0px solid ; width: 400px; height: 400px;" alt="w7" src="https://static.igem.org/mediawiki/2010/5/5f/Br4.jpg"></a></div></html>
 +
<br> According to the SDS-Page result, Ferric Citrate increses the expression of CooA. It is probably releated to heme characteristic of CooA. The best IPTG induction time among 3-5-7-9 hours is 5 hour.
 +
<br>  <html><div><a href="https://2010.igem.org/Team:METU_Turkey/Results_Discussion/Ferric_Citrate_effect"><strong>Details...</strong></a></div></html>
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.</p>
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<br> <h3>E. Soluble versus insoluble fraction</h3>
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</td>
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<br> Lysis Buffer with Urea and without Urea
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</tr>
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</table>  
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</div> <!-- close main item -->
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<div align="center" style="margin:15px 0px 0px 0px"><img style="border: 0px solid ; width: 400px; height: 400px;" alt="w7" src="https://static.igem.org/mediawiki/2010/4/43/Br5.jpg"></a></div></html>
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 +
<br> Results are seen in the SDS-Page photograph, there is no difference between bands of same samples (look at all the 50kDa bands). Thus,we concluded that CooA does not accumulate in inclusion body.
 +
 
 +
<br>  <html><div><a href="https://2010.igem.org/Team:METU_Turkey/Results_Discussion/Soluble_vs_insoluble_fraction"><strong>Details...</strong></a></div></html>
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 +
<br> <h2>2-Purification optimization</h2>
 +
 
 +
<br> <h3>A. Ion exchange chromatography with Sepharose Q (QS)</h3>
 +
 
 +
<br> Purification QS with BL21 pTriEx: Yellowish color indicates our CooA protein. Then we looaded 27,28,29,30 and 31 to SDS-Gel the elutes which have a high value at 420nm because oxidized CooA gives a Sorret peak at 420 nm according to Aono. CooA concentration was determined according to extinction coefficient of heme (Ferric State) which is 108 mM-1 cm-1 according to Aono. The SDS gel indicates that our protein is colose to 50 kDa in dimer. MW of monomer is estimated 24-25kDa according to Aono.
 +
 
 +
 
 +
<br>  <html><div><a href="https://2010.igem.org/Team:METU_Turkey/Results_Discussion/QS_results"><strong>Details...</strong></a></div></html>
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 +
<html>
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<div align="center" style="margin:15px 0px 0px 0px"><img style="border: 0px solid ; width: 400px; height: 400px;" alt="w7" src="https://static.igem.org/mediawiki/2010/1/14/Br13.jpg"></a></div></html>
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<html>
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<div align="center" style="margin:15px 0px 0px 0px"><img style="border: 0px solid ; width: 400px; height: 400px;" alt="w7" src="https://static.igem.org/mediawiki/2010/7/76/B30.jpg"></a></div></html>
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<html>
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<div align="center" style="margin:15px 0px 0px 0px"><img style="border: 0px solid ; width: 400px; height: 400px;" alt="w7" src="https://static.igem.org/mediawiki/2010/9/93/Br14.jpg"></a></div></html>
 +
 
 +
<br> <h3>B. Affinity chromatography with chealating sepharose(CS)</h3>
 +
 
 +
<br> Purification with CS BL21 pTriEx: Loaded combined FPLC tubes from QS column(10/09/26) gave two close picks in CS column. First one corresponds to the CooA. The elutions of CooA are C13,C14 and C15. We combined them for further analyzes with EMSA and ITC and stored @+4. C16 AND C17 are combined to reload to CS column because they are contaminated another protein which is close to 40kDa.
 +
 
 +
 
 +
<br>  <html><div><a href="https://2010.igem.org/Team:METU_Turkey/Results_Discussion/CS"><strong>Details...</strong></a></div></html>
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 +
<html>
 +
<div align="center" style="margin:15px 0px 0px 0px"><img style="border: 0px solid ; width: 400px; height: 400px;" alt="w7" src="https://static.igem.org/mediawiki/2010/7/74/Br15.jpg"></a></div></html>
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<html>
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<div align="center" style="margin:15px 0px 0px 0px"><img style="border: 0px solid ; width: 400px; height: 400px;" alt="w7" src="https://static.igem.org/mediawiki/2010/d/d8/Br16.jpg"></a></div></html>
 +
 
 +
 
 +
<br> <h2>C. Concentrated CooA</h2>
 +
 
 +
<br> CooA protein contains heme group therefore its solution is seen yellow to orange
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 +
<html>
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<div align="center" style="margin:15px 0px 0px 0px"><img style="border: 0px solid ; width: 400px; height: 400px;" alt="w7" src="https://static.igem.org/mediawiki/2010/b/be/Br18.jpg"></a></div></html>
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<br> -- </br>

Latest revision as of 03:39, 28 October 2010

Home

 


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3-CooA Expression and Purification



1-Overexpression optimization


A. Expression vector comparison (pKK versus pTriEx)


When we compare the below results, the expression of pTriEx vector is higher than the expression of pTriEx vector

A.1. Expression in BL21 with pTriEx


In this expression experiment, we obtained 2 different bands in BL21 pTriEx from BL21 negative control as indicated gel photo. CooA is found in two form (monomer and dimer).
w7

A.2. Expression in JM109 and BL21 with pKK vector

We failed to express pKK vector in JM109. We have a little expression in BL21.
w7



Contents

B. Expression host comparison (JM109 versus BL21)


BL21 is better host than JM109 for CooA.


C. IPTG induction studies


Expression comparison with different IPTG Concentration of pTriEx vector.

w7


Our CooA pTriEx vector is expressed without IPTG induction but is less than others. When 2mM IPTG was added to culture, expression of CooA was decreased. It has probably a toxic effect on our bacteria.



D. Ferric Citrate effect


Time Dependent Expression of pTriEx vector with Ferric Citrate and without Ferric Citrate

w7


According to the SDS-Page result, Ferric Citrate increses the expression of CooA. It is probably releated to heme characteristic of CooA. The best IPTG induction time among 3-5-7-9 hours is 5 hour.



E. Soluble versus insoluble fraction


Lysis Buffer with Urea and without Urea

w7


Results are seen in the SDS-Page photograph, there is no difference between bands of same samples (look at all the 50kDa bands). Thus,we concluded that CooA does not accumulate in inclusion body.



2-Purification optimization


A. Ion exchange chromatography with Sepharose Q (QS)


Purification QS with BL21 pTriEx: Yellowish color indicates our CooA protein. Then we looaded 27,28,29,30 and 31 to SDS-Gel the elutes which have a high value at 420nm because oxidized CooA gives a Sorret peak at 420 nm according to Aono. CooA concentration was determined according to extinction coefficient of heme (Ferric State) which is 108 mM-1 cm-1 according to Aono. The SDS gel indicates that our protein is colose to 50 kDa in dimer. MW of monomer is estimated 24-25kDa according to Aono.



w7

w7

w7


B. Affinity chromatography with chealating sepharose(CS)


Purification with CS BL21 pTriEx: Loaded combined FPLC tubes from QS column(10/09/26) gave two close picks in CS column. First one corresponds to the CooA. The elutions of CooA are C13,C14 and C15. We combined them for further analyzes with EMSA and ITC and stored @+4. C16 AND C17 are combined to reload to CS column because they are contaminated another protein which is close to 40kDa.



w7

w7



C. Concentrated CooA


CooA protein contains heme group therefore its solution is seen yellow to orange

w7




-- </br>

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