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This 3 page Class Notes was uploaded by Mathu Karthikeyan on Monday February 29, 2016. The Class Notes belongs to NE 102 at Boston University taught by Pastorino in Spring 2016. Since its upload, it has received 29 views. For similar materials see Introduction to Cellular and Molecular Biology in Neuroscience at Boston University.
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Date Created: 02/29/16
Lab 4 Thursday, February 25, 20162:05 PM Lab 1: PCR Lab 2: PCR Purification/ Digestion Lab 3: Gel Extraction/Ligation Lab 4: Transformation • Transformation Introducing recombinant DNA in to bacteria ○ • Transfection ○ Introducing recombinant DNA in to a mammalian cell • Transduction ○ Introducing DNA into something using a virus instead of a plasmid • Cell Walls of Bacteria ○ Made up of a matrix of peptidoglycan ○ Transpeptidase-allows the cell to make the cell wall (enzyme for cell wall synthesis) ○ Ampicillin ○ Irreversibly blocks transpetidase -->blocking cell wall synthesis • Parts of the Plasmid ○ MCS-where restriction enzymes can target and cut the plasmid open for the gene of interest ○ Antibiotic resistant gene ○ Origin of replication • Assignment #3 ○ At the C-terminal end there is a series of amino acids (SLL) ○ The LL part of it helps traffic for intracellular localization ○ The S (serine) is a phosphorylation site that modulates LL trafficking This process allows certain transmembrane proteins to come ○ inside the cell ○ Mutation ( the BACE is the same but the 3 amino acids at the end are changed) § Change LL to AA § Change S to A (serine to alanine) ○ C-Transfection inside the cell ○ Mutation ( the BACE is the same but the 3 amino acids at the end are changed) § Change LL to AA § Change S to A (serine to alanine) ○ C-Transfection § Insert into a mammalian cell □ BACE WT (with myc tag or V5) □ Either the BACE-SAA or BACE-ALL § Indirect Immunofluorescence □ WT BACE: Green ® Located around the nucleus □ Mutated BACE: RED ® Mostly near the membrane □ There is no c-localization with BESAA ® The WT BACE was able to enter the cell □ There is localization with BALL ® S is not responsible for internalizing BACE □ LL-may regulate BACE internalization □ S-NOT needed for BACE internalization § Compare and Uantify Levels of BACE at Surface vs Total □ Surface BACE levels: LL/AA >>WT □ Total BACE levels: LL/AA>>WT □ Why isn't Total BACE LL/AA=WT? ® Internalized BACE might be degrading (dying) ® Follow up study: The internalized BACE may be degraded vi lysosomes § Figure 2 □ BACE localization over time ® Unpermeabilized ◊ Antibodies can’t attached to cells inside the cell ® Permeabilized ◊ Antibodies can attach to cells both inside and outside the cell □ WT ® Starts at the membrane and after 45 mins, it is near the nucleus □ LL/AA ® Starts at the plasma membrane and remains at the plasma membrane □ SA ® Starts in the plasma membrane then travels inside and ends up in the same small structures as they did in WT ® Starts at the plasma membrane and remains at the plasma membrane □ SA ® Starts in the plasma membrane then travels inside and ends up in the same small structures as they did in WT § TAKE HOME □ LL motif is responsible for internalizing BACE □ Internalized BACE can be degraded