Difference between revisions of "Part:BBa K3788003:Experience"
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<h2> PCR amplification of 6his<i>p20</i>_flag<i>Cry11Aa</i> genes: </h2> | <h2> PCR amplification of 6his<i>p20</i>_flag<i>Cry11Aa</i> genes: </h2> | ||
| − | <p> The DNA sequence of flag<i>cry11Aa</i> and 6his<i>p20</i> | + | <p> The DNA sequence of flag<i>cry11Aa</i> and 6his<i>p20</i> was amplified by PCR Q5 to generate fragment allowing a SLIC cloning. The expected size is <b>2540bp </b> and a 2000bp band is observed . Amplification of DNAs sequences with primers was validated.</p> |
https://2021.igem.org/wiki/images/a/a3/T--Aix-Marseille--DNA-SLIC-Amplification-xtoxtox.png | https://2021.igem.org/wiki/images/a/a3/T--Aix-Marseille--DNA-SLIC-Amplification-xtoxtox.png | ||
| − | <p><u>Figure</u>. <b>Verification PCR amplification of DNAs sequence and these extensions for SLIC cloning.</b> <i> | + | <p><u>Figure 1</u>. <b>Verification PCR amplification of DNAs sequence and these extensions for SLIC cloning.</b> <i> |
a. Control – (Absence of DNAs, no band expected); b, c, d, e. Amplification of 6hisp20_flagcry11Aa (expected 2500 bp), flagcry11Aa (expected 1847 bp), strepcyt1Aa (expected 775 bp) and 6hisp20 (expected 568 bp); L (SMART Ladder, its size is shown on the right side). </i></p> | a. Control – (Absence of DNAs, no band expected); b, c, d, e. Amplification of 6hisp20_flagcry11Aa (expected 2500 bp), flagcry11Aa (expected 1847 bp), strepcyt1Aa (expected 775 bp) and 6hisp20 (expected 568 bp); L (SMART Ladder, its size is shown on the right side). </i></p> | ||
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<p><h2> Expression of Flag-Cry11Aa and 6his-P20 : </h2></p> | <p><h2> Expression of Flag-Cry11Aa and 6his-P20 : </h2></p> | ||
| − | <p> The 6his<i>p20</i>_flag<i>Cry11Aa</i> genes were | + | <p> The 6his<i>p20</i>_flag<i>Cry11Aa</i> genes were cloned into pBAD24 plasmid, genes are under the control of Arabinosis. An expression test was done to see if P20 and Cry11Aa are produced in <i>E. coli</i> and if toxins have the expected size (<b>20 and 70 kDa</b>). To do it, the toxin Cry11Aa have a <b>flag tag in Nter</b> and P20 chaperon have a <b>6x His tag in Nter</b> , it allows to have a great detection by Western Blot. </p> |
<p><b>P20 and Cry11Aa are expressed and have expected sizes.</b></p> | <p><b>P20 and Cry11Aa are expressed and have expected sizes.</b></p> | ||
https://2021.igem.org/wiki/images/3/32/T--Aix-Marseille--P20-Cry11Aa-expression.png | https://2021.igem.org/wiki/images/3/32/T--Aix-Marseille--P20-Cry11Aa-expression.png | ||
| − | <p><u>Figure</u>. <b>Proteins’ expressions</b>. Flag-Cry11Aa and 6his-P20 were expressed by MG1655 E. coli strain hold pBAD24_6hisp20_flagcry11Aa plasmid. When bacteria were in exponential phase (OD600nm = 0.4), pBAD24 was induced with Arabinoses 0.1%. E. coli is grown at 37°C stirring in Luria-Bertani Ampicillin medium. These proteins were revealed by Westernblot using antibody antibody anti-histidine or antibody anti flag tag. Molecular size markers are indicated in kDa “RulePAGE Ladder". </p> | + | <p><u>Figure 2</u>. <b>Proteins’ expressions</b>. Flag-Cry11Aa and 6his-P20 were expressed by MG1655 E. coli strain hold pBAD24_6hisp20_flagcry11Aa plasmid. When bacteria were in exponential phase (OD600nm = 0.4), pBAD24 was induced with Arabinoses 0.1%. E. coli is grown at 37°C stirring in Luria-Bertani Ampicillin medium. These proteins were revealed by Westernblot using antibody antibody anti-histidine or antibody anti flag tag. Molecular size markers are indicated in kDa “RulePAGE Ladder". </p> |
<p>pBAD24_6hisp20_flagcry11Aa ino induced (Ni) and induced at different times samples (2H, 3H or 4H). The expected size for P20 is 20kDa and 70kDa is the expected size for Cry11Aa.</p> | <p>pBAD24_6hisp20_flagcry11Aa ino induced (Ni) and induced at different times samples (2H, 3H or 4H). The expected size for P20 is 20kDa and 70kDa is the expected size for Cry11Aa.</p> | ||
Latest revision as of 09:13, 21 October 2021
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Applications of BBa_K3788003
PCR amplification of 6hisp20_flagCry11Aa genes:
The DNA sequence of flagcry11Aa and 6hisp20 was amplified by PCR Q5 to generate fragment allowing a SLIC cloning. The expected size is 2540bp and a 2000bp band is observed . Amplification of DNAs sequences with primers was validated.
Figure 1. Verification PCR amplification of DNAs sequence and these extensions for SLIC cloning. a. Control – (Absence of DNAs, no band expected); b, c, d, e. Amplification of 6hisp20_flagcry11Aa (expected 2500 bp), flagcry11Aa (expected 1847 bp), strepcyt1Aa (expected 775 bp) and 6hisp20 (expected 568 bp); L (SMART Ladder, its size is shown on the right side).
Expression of Flag-Cry11Aa and 6his-P20 :
The 6hisp20_flagCry11Aa genes were cloned into pBAD24 plasmid, genes are under the control of Arabinosis. An expression test was done to see if P20 and Cry11Aa are produced in E. coli and if toxins have the expected size (20 and 70 kDa). To do it, the toxin Cry11Aa have a flag tag in Nter and P20 chaperon have a 6x His tag in Nter , it allows to have a great detection by Western Blot.
P20 and Cry11Aa are expressed and have expected sizes.
Figure 2. Proteins’ expressions. Flag-Cry11Aa and 6his-P20 were expressed by MG1655 E. coli strain hold pBAD24_6hisp20_flagcry11Aa plasmid. When bacteria were in exponential phase (OD600nm = 0.4), pBAD24 was induced with Arabinoses 0.1%. E. coli is grown at 37°C stirring in Luria-Bertani Ampicillin medium. These proteins were revealed by Westernblot using antibody antibody anti-histidine or antibody anti flag tag. Molecular size markers are indicated in kDa “RulePAGE Ladder".
pBAD24_6hisp20_flagcry11Aa ino induced (Ni) and induced at different times samples (2H, 3H or 4H). The expected size for P20 is 20kDa and 70kDa is the expected size for Cry11Aa.
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