Difference between revisions of "Part:BBa K4632003:Experience"

 
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===Applications of BBa_K4632003===
 
===Applications of BBa_K4632003===
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<p><strong> 1. Verifying the Expression and Secretion Proficiency of CPTI</strong></p>
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We ordered the OMPA-CPTI fragment from GUANGZHOU IGE BIOTECHNOLOGY LTD and inserted it into the pET30a vector.
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[https://static.igem.wiki/teams/4632/wiki/wiki/registry-part/pet30a-ompa-cpti-part-2-1.png]
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Then, we transformed pET30a-OMPA-CPTI into ''E. coli'' BL21, induced expression with IPTG for 3 hours, and then performed centrifugation to obtain the supernatant and pellet fractions.
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''https://static.igem.wiki/teams/4632/wiki/wiki/registry-part/part-3-1-1-1.png''
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<p><strong> Figure1: </strong> Diagram of CPTI circuit design</p>
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<p>The supernatant was concentrated using ultrafiltration centrifuge tubes, while the pellet was subjected to disruption with lysis buffer. After disruption, we centrifuged the sample to separate the supernatant from the disrupted pellet, and the disrupted pellet was resuspended in lysis buffer.</p>
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Since 12% SDS-PAGE did not adequately display the expression results, we also attempted Tricine-PAGE and increased the SDS-PAGE separation gel concentration to 17.5%. However, upon analyzing these samples using Tricine-PAGE and Western Blot, the results revealed that the CPTI protein was not expressed in ''E. coli'' (as shown in Figures 2 and 3).
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"https://static.igem.wiki/teams/4632/wiki/wiki/registry-part/part-2-5.png"
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<p><strong> Figure 2: </strong>Tricine-PAGE analysis of CPTI expression
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Lane 1：Concentrated supernatant of OmpA-CPTI (+IPTG); Lane 2: Concentrated supernatant of OmpA-CPTI (-IPTG); Lane 3: Concentrated supernatant of pET-30a(+IPTG); Lane4:Whole cell lysate of OmpA-CPTI (+IPTG);Lane5:Whole cell lysate of OmpA-CPTI (-IPTG);Lane6:Whole cell lysate of CPTI (+IPTG);Lane7:Whole cell lysateof of CPTI(-IPTG);Lane8:Whole cell lysate of pET-30a(+IPTG);Lane9:Whole cell lysate of pET-30a(-IPTG)
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</p>
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''https://static.igem.wiki/teams/4632/wiki/wiki/registry-part/part-2-6.png''
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<p><strong> Figure 3: </strong>Western blot analysis of CPTI expression
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Lane 1：Concentrated supernatant of OmpA-CPTI (+IPTG); Lane 2: Concentrated supernatant of OmpA-CPTI (-IPTG); Lane 3: Concentrated supernatant of pET-30a(+IPTG); Lane4:Whole cell lysate of OmpA-CPTI (+IPTG);Lane5:Whole cell lysate of OmpA-CPTI (-IPTG);Lane6:Whole cell lysate of CPTI (+IPTG);Lane7:Whole cell lysate of of CPTI(-IPTG);Lane8:Whole cell lysate of pET-30a(+IPTG);Lane9:Whole cell lysate of pET-30a(-IPTG)</p>
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    <p><strong>In conclusion</strong>, the expression of the CPTI protein in ''E. coli'' was unsuccessful as observed in the results from both 12% SDS-PAGE, Tricine-PAGE, and 17.5% SDS-PAGE. Further investigation and optimization may be necessary to achieve successful protein expression.</p>
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      <p><strong>Subsequently</strong>, we have tried to constructed the CPTI gene with a GST tag[https://static.igem.wiki/teams/4632/wiki/wiki/registry-part/part-2-4.png], and protein induction expression is currently underway. This modification is intended to ensure that the protein is expressed to a significant extent. Previous literature has demonstrated a similar approach, and the key difference in our attempt is to test the activity of CPTI without removing the GST tag. </p>
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===User Reviews===
 
===User Reviews===

Revision as of 15:17, 10 October 2023


This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K4632003

1. Verifying the Expression and Secretion Proficiency of CPTI


We ordered the OMPA-CPTI fragment from GUANGZHOU IGE BIOTECHNOLOGY LTD and inserted it into the pET30a vector. [1] Then, we transformed pET30a-OMPA-CPTI into E. coli BL21, induced expression with IPTG for 3 hours, and then performed centrifugation to obtain the supernatant and pellet fractions.

part-3-1-1-1.png

Figure1: Diagram of CPTI circuit design

The supernatant was concentrated using ultrafiltration centrifuge tubes, while the pellet was subjected to disruption with lysis buffer. After disruption, we centrifuged the sample to separate the supernatant from the disrupted pellet, and the disrupted pellet was resuspended in lysis buffer.

Since 12% SDS-PAGE did not adequately display the expression results, we also attempted Tricine-PAGE and increased the SDS-PAGE separation gel concentration to 17.5%. However, upon analyzing these samples using Tricine-PAGE and Western Blot, the results revealed that the CPTI protein was not expressed in E. coli (as shown in Figures 2 and 3).


"part-2-5.png"

Figure 2: Tricine-PAGE analysis of CPTI expression Lane 1：Concentrated supernatant of OmpA-CPTI (+IPTG); Lane 2: Concentrated supernatant of OmpA-CPTI (-IPTG); Lane 3: Concentrated supernatant of pET-30a(+IPTG); Lane4:Whole cell lysate of OmpA-CPTI (+IPTG);Lane5:Whole cell lysate of OmpA-CPTI (-IPTG);Lane6:Whole cell lysate of CPTI (+IPTG);Lane7:Whole cell lysateof of CPTI(-IPTG);Lane8:Whole cell lysate of pET-30a(+IPTG);Lane9:Whole cell lysate of pET-30a(-IPTG)


part-2-6.png

Figure 3: Western blot analysis of CPTI expression Lane 1：Concentrated supernatant of OmpA-CPTI (+IPTG); Lane 2: Concentrated supernatant of OmpA-CPTI (-IPTG); Lane 3: Concentrated supernatant of pET-30a(+IPTG); Lane4:Whole cell lysate of OmpA-CPTI (+IPTG);Lane5:Whole cell lysate of OmpA-CPTI (-IPTG);Lane6:Whole cell lysate of CPTI (+IPTG);Lane7:Whole cell lysate of of CPTI(-IPTG);Lane8:Whole cell lysate of pET-30a(+IPTG);Lane9:Whole cell lysate of pET-30a(-IPTG)


In conclusion, the expression of the CPTI protein in E. coli was unsuccessful as observed in the results from both 12% SDS-PAGE, Tricine-PAGE, and 17.5% SDS-PAGE. Further investigation and optimization may be necessary to achieve successful protein expression.

Subsequently, we have tried to constructed the CPTI gene with a GST tag[2], and protein induction expression is currently underway. This modification is intended to ensure that the protein is expressed to a significant extent. Previous literature has demonstrated a similar approach, and the key difference in our attempt is to test the activity of CPTI without removing the GST tag.



User Reviews

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