lab 6 DGD - BCH 3356 English Thursday 8:30-10:00 TBT333...

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BCH 3356 English – Thursday 8:30-10:00 TBT333 English – Friday 2:30-4:00 MNT203 Megan Rose –
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Lab 5 Reinoculation of the positive transformant cell line (selected during lab 4) into a liquid culture and IPTG- induction for overexpression of T7 RNA polymerase. AMP IPTG
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After 1.5hr of induction, we will observe an over- representation of our T7 RNA polymerase fusion protein in the IPTG-induced aliquot. The water-induced aliquot at 1.5 hr is the adequate negative control for the IPTG- induced aliquot (both cultures were grown for an extra 1.5hr). Both proteomes should be Lab 5 - controls T = 1.5h W I Induced with… W = water I = IPTG
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Leaky expression A small quantity of recombinant proteins will be produced from the leaky expression in the negative control (no IPTG) Remember: In the absence of lactose, the lacIq repressor is bound tightly to the operon promoter repressing gene expression However, once the glucose levels have depleted the cell will need to express the genes necessary for lactose
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Leaky expression Remember: In the presence of lactose or an analog (IPTG), the lac promoter is de-repressed and transcription is allowed (the inducer binds the repressor and stops it from binding to the lac operator). When glucose levels start decreasing, the E.coli bacteria will start using lactose as a source of energy and a small de- repression of the trp-lac promoter will be observed.
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The control with the empty vector illustrates the effect of IPTG on the E. coli proteome Since the T7 insert is absent in these cells, we will have a protein extract representing the E.coli proteome induced by the IPTG. The T7 RNA polymerase fusion protein would be completely absent in this control – only the his- tag will be expressed. Lab 5 - controls 0h 1.5 h
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Immobilized Metal ion Affinity Chromatography (IMAC) Molecular principal: Formation of a bond between an immobilized metal ion (in this case Ni 2+ ) and an electron donor present on the recombinant protein to be purified (in this case the nitrogen from the imidazole ring of
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Purification of His-tagged proteins Histidine residues have a strong affinity for metal ions and are not usually present on protein surfaces, this makes them ideal candidates for purification by IMAC. We use vectors that are genetically engineered
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proteins Imidazole is used to elute recombinant his- tagged proteins bound to the nickel ion. An excess of imidazole
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lab 6 DGD - BCH 3356 English Thursday 8:30-10:00 TBT333...

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