Team:Trieste/notebook3

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Week 3

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Suicide System

We inoculated the positive colonies and also checked by digestion ( EcoRI and XbaI/PstI ). The LL37 fragment, cut XbaI/PstI, was successfully cloned downstream the promoter T5LacOperator cut SpeI/PstI. The M15 cells ( containing the pREP4 plasmid coding for the Lac repressor) were transformed with the construct described above and seeded in Kan ( M15 resistance), Cm ( pSB1C3 resistance) in two conditions non induced and induced with IPTG ( the inhibitors of the repressor Lac).

Unfortunately the LL-37 cathelicidin was probably unable to disrupt the cell membrane, as we observed a growth both in induced and not induced.

The construct T5CumateOperator-LL37 was cut EcoRI/XbaI, heat inactivated at 80∞C for 20í, dephosphorilated at its extremitis.

Antibody

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Chassis

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