Announcements Quiz Pre lab Lecture Review so far colony data Genetic control elements Sequencing recap Today in Lab Mod 2 Day 5 Announcements No quiz next time full day Responsible for Day 5 6 material for Day 7 quiz Module 1 revision due by 11 am next time Previous quiz and FNT ctrl exp don t work Master Mix cycler ctrl worked exp didn t primers DNA template mutagenize pRSET IPC pRSET IPC DAY 2 liquid culture DAY 3 miniprep mutant DNA DAY 4 liquid culture transform in XL1 Blue induce cells with IPTG DAY 5 transform in BL21 DE3 DAY 4 lyse cells and extract protein DAY 6 evalute protein DAY 7 8 Colony counts Teaching faculty plates pWhitescript 600 M124S 1 300 225 M124S 1 400 52 lac operon These three genes encode metabolic enzymes lacI control lacZ lacY lacA promoter operator Encodes a repressor protein that binds to control area turning it OFF In turn if lactose binds to the repressor it is made inactive turning ON expression of Z Y A Induction of a chosen protein lacI control T7RNAP polymerase lacZ T7RNAP gene is expressed in presence of lactose or analogue IPTG 0 IPTG t bla promoter is constitutively on T7 promoter is turned on in presence of T7RNAP BL21 DE3 bacterial strain me o n ge DE3 bacteriophage virus used to integrate lac T7RNAP into E coli mi d s a l p pLysS protein that produces lysozyme which binds to T7RNAP reducing leaky expression Retained by chloramphenicol selection Sequencing reactions Dideoxy method no 3 OH can t elongate Run 4 rxns d dT dA dG dC and 3 others radioactive or fluorescent label different fluorophore ddA A C T Today in Lab Samples Start with four DE3 samples carrying plasmid WT M124S X Z candidates 1 and 2 After gel and sequencing analysis pick just one X Z to continue working with End of day hand in 6 pellets or 3 pellets 3 cultures and 3 eppendorfs to teaching faculty MIT OpenCourseWare http ocw mit edu 20 109 Laboratory Fundamentals in Biological Engineering Spring 2010 For information about citing these materials or our Terms of Use visit http ocw mit edu terms
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