Difference between revisions of "Part:BBa K3788000:Experience"

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Applications of BBa_K3788000

PCR amplification of strepcyt1Aa gene :

The DNA sequence of strepcyt1Aa was amplified by PCR Q5 to generate fragment allowing a SLIC cloning. The expected size is 775 bp and a 800bp 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 Strep-Cyt1Aa :

The strepcyt1Aa gene was cloned into pBAD24 plasmid, gene are under the control of Arabinosis. An expression test was done to see if the Cyt1Aa toxin is produced in E. coli and if the toxin have the expected size (28kDa). To do it, the toxins have a Strep tag II in Nter, it allows to have a great detection by Western Blot.

Strep-Cyt1Aa is expressed and have the expected size.

Figure 2. Proteins’ expressions. Strep-Cyt1Aa was expressed by MG1655 E. coli strain hold pBAD24_strepcyt1Aa 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. This protein was revealed by Westernblot using antibody anti-strep II. Molecular size markers are indicated in kDa “RulePAGE Ladder”.

pBAD24_strepcyt1Aa induced or no induced sample. 1UDO was taken after 4 H of induction. The expected size for Cyt1Aa is 28kDa. T+ Strep is a positive control of antibodies

Strep-Cyt1Aa purification :

We tried to purify Strep-Cyt1Aa using a Strep tag II column affinity (after the Strep-Cyt1Aa overproduction). Are visible bands at 25 kDa corresponding to every sample (except the wash). The positive antibody control is also visible. The expected size for Strep-Cyt1Aa is 28kDa. Finally, bands visible are bands expected.

We are able to purify Strep-Cyt1Aa

Figure 3. Strep-Cyt1Aa’s purification. Strep-Cyt1Aa toxins were expressed by MG1655 E. coli strain hold pBAD24_strepcyt1Aa plasmid. When bacteria were in exponential phase (OD600nm = 0.4), pBAD24 was induced with Arabinoses 1%. E. coli grown at 28°C stirring, overnight in 2 litters of Luria-Bertani.

a) Strep-Cyt1Aa expressed was revealed by Westernblot using antibody antibody anti-strep II. Molecular size markers are indicated in kDa “RulePAGE Ladder”. The “No induced”, “total fraction”, “soluble part” and “unsoluble part” samples represent, respectively, MG1655 E. coli strain holding pBAD24_strepcyt1Aa plasmid no induced by arabinose, induced part holding 1UDO of bacteria, the cytoplasmic material after lysis and the membrane material solubilised. A positive sample of antibody anti-strep was deposited on the gel for the Westernblot

b) Strep-Cyt1Aa toxins were purified thanks to a <b>affinity chromatography strep-tactin column and revealed by Westernblot using antibody antibody anti-strep II . Molecular size markers are indicated in kDa “RulePAGE Ladder”. The “No induced”, “total fraction”, “soluble part” and “unsoluble part” samples represent, respectively, MG1655 E. coli strain holding pBAD24_strepcyt1Aa plasmid no induced by arabinose, induced part holding 1UDO of bacteria, the cytoplasmic material after lysis and the membrane material solubilized. A positive sample of antibody anti-strep was deposited on the gel for the Westernblot

The expected size is 28kDa.

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