Difference between revisions of "Part:BBa K4165256"
(→WetLab Results) |
|||
Line 13: | Line 13: | ||
===WetLab Results=== | ===WetLab Results=== | ||
+ | In the wet lab we started with cloning in the pJET vector followed by the expression in the pgs21a, then we performed two different kinds of lysis to extract the protein to find which lysis buffer will give better yield, and quantified the protein expression before and after induction using BCA assay, in the end, we tested the GST COH WWW affinity by the pulldown assay against the His Trim21 (L) DOC and against the tau aggregates which showed that WWW could bind to the tau aggregates effectively | ||
<p style=" font-weight: bold; font-size:14px;"> Transformation of GST COH WWW in BL-21 using pGS-21a vector </p> | <p style=" font-weight: bold; font-size:14px;"> Transformation of GST COH WWW in BL-21 using pGS-21a vector </p> | ||
+ | The transformation was done using TSS buffer protocol, after trying three buffers which are Calcium chloride, Magnesium chloride and a combination between Calcium chloride and Magnesium chloride, we optimized our protocol to use the TSS buffer protocol as it showed the best results with a transformation efficiency of GST COH WWW in DH-5 alpha using pJET vector is 14200000 no. of transformants/µg while that of GST COH WWW in BL-21 using pGS-21a vector is 700 no.of transformants/µg , you can find the complete protocol in our wiki page | ||
<html> | <html> | ||
<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/wetlab-results/gst-coh-www-pgs.jpg" style="margin-left:200px;" alt="" width="500" /></p> | <p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/wetlab-results/gst-coh-www-pgs.jpg" style="margin-left:200px;" alt="" width="500" /></p> | ||
Line 24: | Line 26: | ||
Figure 2. Transformed plate of GST COH (L) WWW + pJET | Figure 2. Transformed plate of GST COH (L) WWW + pJET | ||
<p style=" font-weight: bold; font-size:14px;"> Comparison between chemical lysis and sonication for GST COH WWW </p> | <p style=" font-weight: bold; font-size:14px;"> Comparison between chemical lysis and sonication for GST COH WWW </p> | ||
+ | Chemical lysis and sonication were done to check which of them gives better results in the protein extraction, and after comparing the results we optimized our protocol to use chemical lysis for GST COH WWW | ||
<html> | <html> | ||
<p><img src="https://static.igem.wiki/teams/4165/wiki/data-analysis/sonication-or-chemical/sonication-or-chemical/coh-www.jpg" style="margin-left:200px;" alt="" width="500" /></p> | <p><img src="https://static.igem.wiki/teams/4165/wiki/data-analysis/sonication-or-chemical/sonication-or-chemical/coh-www.jpg" style="margin-left:200px;" alt="" width="500" /></p> | ||
Line 30: | Line 33: | ||
WWW, after we had the results we optimized our protocol to use chemical lysis for GST COH WWW | WWW, after we had the results we optimized our protocol to use chemical lysis for GST COH WWW | ||
<p style=" font-weight: bold; font-size:14px;"> SDS PAGE for induced and non induced samples of GST COH WWW </p> | <p style=" font-weight: bold; font-size:14px;"> SDS PAGE for induced and non induced samples of GST COH WWW </p> | ||
+ | SDS PAGE depends on the molecular weight of the protein, we performed SDS to make sure that our protein is in the exact size and to show the difference between induced and non induced samples of GST COH WWW. | ||
<html> | <html> | ||
<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/wetlab-results/sds-of-gst-td28rev-and-www.jpg" style="margin-left:200px;" alt="" width="500" /></p> | <p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/wetlab-results/sds-of-gst-td28rev-and-www.jpg" style="margin-left:200px;" alt="" width="500" /></p> | ||
Line 37: | Line 41: | ||
effectively owing to our right choice of IPTC, time interval and concentration | effectively owing to our right choice of IPTC, time interval and concentration | ||
<p style=" font-weight: bold; font-size:14px;"> Pull down assay of His Trim21 (L) against GST COH WWW and GST COH TD28Rev </p> | <p style=" font-weight: bold; font-size:14px;"> Pull down assay of His Trim21 (L) against GST COH WWW and GST COH TD28Rev </p> | ||
+ | Pull down assay is a technique performed to check the interactions between the proteins and to check if they bind properly, we performed pull down assay to check the binding between GST COH WWW against His Trim21 (L) DOC, and beyween Tau aggregates and GST COH WWW, where WWW was found to be a proper candidate of Tau (Illustrated in figure 5 and 6) | ||
<html> | <html> | ||
<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/wetlab-results/pull-down-trim-doc-vs-www-and-td28rev.jpg" style="margin-left:200px;" alt="" width="500" /></p> | <p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/wetlab-results/pull-down-trim-doc-vs-www-and-td28rev.jpg" style="margin-left:200px;" alt="" width="500" /></p> | ||
Line 54: | Line 59: | ||
binding to the Tau aggregates | binding to the Tau aggregates | ||
<p style=" font-weight: bold; font-size:14px;"> BCA assay results of GST COH WWW </p> | <p style=" font-weight: bold; font-size:14px;"> BCA assay results of GST COH WWW </p> | ||
+ | BCA assay is a technique that is performed to quantify the proteins, and it depends on the color of the BCA dye which is directly proportional with the quantity of the protein, we performed BCA for GST COH WWW to know its concentration and it is found to be 0.719466667 | ||
<html> | <html> | ||
<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/wetlab-results/standard-curve.jpg" style="margin-left:200px;" alt="" width="500" /></p> | <p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/wetlab-results/standard-curve.jpg" style="margin-left:200px;" alt="" width="500" /></p> | ||
Line 61: | Line 67: | ||
</html> | </html> | ||
Figure 7. This graph illustrates the results of BCA assay for GST COH WWW showing that our protein | Figure 7. This graph illustrates the results of BCA assay for GST COH WWW showing that our protein | ||
− | concentration is expected to be 0.719466667 | + | concentration is expected to be 0.719466667 |
− | + | ||
<!-- Uncomment this to enable Functional Parameter display | <!-- Uncomment this to enable Functional Parameter display | ||
===Functional Parameters=== | ===Functional Parameters=== | ||
<partinfo>BBa_K4165256 parameters</partinfo> | <partinfo>BBa_K4165256 parameters</partinfo> | ||
<!-- --> | <!-- --> |
Revision as of 14:17, 12 October 2022
GST-Coh2-G4S-WWW
Long description
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 85
WetLab Results
In the wet lab we started with cloning in the pJET vector followed by the expression in the pgs21a, then we performed two different kinds of lysis to extract the protein to find which lysis buffer will give better yield, and quantified the protein expression before and after induction using BCA assay, in the end, we tested the GST COH WWW affinity by the pulldown assay against the His Trim21 (L) DOC and against the tau aggregates which showed that WWW could bind to the tau aggregates effectively
Transformation of GST COH WWW in BL-21 using pGS-21a vector
The transformation was done using TSS buffer protocol, after trying three buffers which are Calcium chloride, Magnesium chloride and a combination between Calcium chloride and Magnesium chloride, we optimized our protocol to use the TSS buffer protocol as it showed the best results with a transformation efficiency of GST COH WWW in DH-5 alpha using pJET vector is 14200000 no. of transformants/µg while that of GST COH WWW in BL-21 using pGS-21a vector is 700 no.of transformants/µg , you can find the complete protocol in our wiki page
Figure 1. Transformed plate of GST COH (L) WWW + pGS-21a
Transformation of GST COH WWW in DH-5 alpha using pJET vector
Figure 2. Transformed plate of GST COH (L) WWW + pJET
Comparison between chemical lysis and sonication for GST COH WWW
Chemical lysis and sonication were done to check which of them gives better results in the protein extraction, and after comparing the results we optimized our protocol to use chemical lysis for GST COH WWW
Figure 3. This graph shows a significant difference between chemical lysis and sonication for GST COH WWW, after we had the results we optimized our protocol to use chemical lysis for GST COH WWW
SDS PAGE for induced and non induced samples of GST COH WWW
SDS PAGE depends on the molecular weight of the protein, we performed SDS to make sure that our protein is in the exact size and to show the difference between induced and non induced samples of GST COH WWW.
Figure 4. This figure shows the comparison between induced and non induced samples of GST COH WWW, where well no.1 is the induced sample while well no.5 is the non induced sample showing that our protein is induced effectively owing to our right choice of IPTC, time interval and concentration
Pull down assay of His Trim21 (L) against GST COH WWW and GST COH TD28Rev
Pull down assay is a technique performed to check the interactions between the proteins and to check if they bind properly, we performed pull down assay to check the binding between GST COH WWW against His Trim21 (L) DOC, and beyween Tau aggregates and GST COH WWW, where WWW was found to be a proper candidate of Tau (Illustrated in figure 5 and 6)
Figure 5. This graph shows the comparison of pull down assay between His trim21(L) DOC with GST COH WWW and GST COH TD28Rev, showing that the interaction between His Trim21 (L) DOC and GST COH WWW is better than that of His Trim21 (L) DOC and GST COH TD28Rev as the concentration of the elution of His Trim21 (L) DOC with GST COH WWW is more than that of His Trim21 (L) DOC with GST COH TD28Rev
Pull down assay of Tau aggregates against GST COH WWW and GST COH TD28Rev
Figure 6. This graph shows the comparison of pull down assay between Tau aggregates with GST COH WWW and GST COH TD28Rev, showing that the interaction between Tau aggregates with GST COH WWW is better than that of Tau aggregates with GST COH TD28Rev as the concentration of elution of Tau aggregates with GST COH WWW is more than that of Tau aggregates with GST COH TD28Rev, illustrating that WWW could be a better candidate for binding to the Tau aggregates
BCA assay results of GST COH WWW
BCA assay is a technique that is performed to quantify the proteins, and it depends on the color of the BCA dye which is directly proportional with the quantity of the protein, we performed BCA for GST COH WWW to know its concentration and it is found to be 0.719466667
Figure 7. This graph illustrates the results of BCA assay for GST COH WWW showing that our protein concentration is expected to be 0.719466667