Part:BBa_K3788000:Experience

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PCR amplification of strepcyt1Aa gene :

The DNA sequence of strepcyt1Aa was amplify by PCR Q5 to generate fragment allowing a SLIC cloning. The expected size is 775 pb and a 800pb band is observed. Amplification of DNAs sequences with primers were validated.

Figure. 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 pB), flagcry11Aa (expected 1847 pB), strepcyt1Aa (expected 775 pB) and 6hisp20 (expected 568 pB); L (SMART Ladder, its size is shown on the right side).

Expression of Strep-Cyt1Aa :

The strepcyt1Aa gene was clone 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 doing it, the toxin have a Strep tag II in Nter, it allow to have a great detection by Western Blot.

Strep-Cyt1Aa is expressed and have the expected size.

Figure. Proteins’ expressions. Strep-Cyt1Aa, Flag-Cry11Aa and 6his-P20 were expressed by MG1655 E. coli strain hold pBAD24_strepcyt1Aa, pBAD24_6hisp20 and pBAD24_6hisp20_flagcry11Aa plasmids. 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 anti-strep II (a), antibody anti-histidine (b, c.) or antibody anti flag tag (c.). 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

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