QBM Exam Questions And Answers With Complete Solutions 2024

QBM Exam Questions And Answers With Complete Solutions 2024 What are the first three steps for starting a project? - answer1 ) Choose 2) Read literature 3) Obtain DNA sequence Once you obtain the DNA sequence in the beginning of a project, BLAST will allow what? - answer"Basic Local Alignment Search Tool" - To search for homology (duplication events) - Can predict protein, molecular weight, pI, and extinction coefficient - Predict the secondary/tertiary structure, transmembrane helices, ubiquitation sites, etc Limitation of synthesizing proteins organic chemistry-wise? - answerLimited to 50-70 amino acids - May have issues with folding (tertiary structure)/post-translational modifications PROCESS: block N-terminus, add C-terminus, block, add, block ,add - takes a while - "simplest method however" Recombinant DNA - answerINsert human gene of interest into a bacterial plasmid to make a *vector/construct* Plasmid can be transformed into bacteria to be expressed Solid phase peptide synthesis does not allow for... - answerPost-translational modifications In vitro expression of protein - answer"Cell free" - Fast (just ribosome and mRNA (with some elognation factors/tRNA) But expensive and *low yield (mg/micrograms)* Benefits of using prokaryotic cells for protein expression? - answer- High levels of expression - Mastered technique (mostly E. coli) - Can express proteins toxic to other systems - Bacteria is cheap, grows quickly - High amount of protein (*grams*) - Clone carrying gene of interest can be selected via blue/white or antibiotics What are some disadvantages of using prokaryotic cells? - answer- May form inclusion bodies when the protein does not fold properly - May not be properly processed (modified - glycoslyated, phosphorylated, etc) - Some proteins toxic to bacteria What are some methods to avoid inclusion bodies? - answer1) Expressed at lower temperatures (20 vs 37C) 2) Modified at sequence level 3) Fused with other proteins to increase solubility/prevent inclusion body formation Eukaryotic systems to express proteins - answerYeast cells/Plant cells - Produce large amounts of protein and fold properly/necessary post-transcriptional modifications (disulfide bond formation, oligomerization, clevage) - Can express multiple genes - Limitless protein size (up to 5000 amino acids) Harder to work with Includes baculovirus Insect cells to express proteins - answerUses baculovirus to infect insect cells more epensive and more involved Mammalian cells as protein expressors - answer- Take large amounts of animal organs and purify proteins directly - Slow - hard to keep alive - Expensive with - Low yield - WIll get final protein with proper modifications - that is, in its *native state* Bacteria are poor compared to mammalian cells when it comes to glycosylation/folding of expressed proteins - answerTrue Insulin expression in prokaryotic vs eukaryotic systems - answerE. coli - requires expression, lysis, purificaiton, and refolding to get it S. cervisiae - expresses straight into supernatent What makes E. coli so widely used in a lab? - answer- Takes up foreign DNA easily - Grows fast, easy to regualte - *Lacks most proteases* - lack recombination What are some example E. coli cell line strains? - answer1) *BL21* = contain T7 RNA polymerase gene 2) *B834* = methionine auxotrophs 3) pLysS = contains plasmid pLysS that expresses T7 lysozyme to inhibit basal levels of expression (toxic compounds) 4) Tuner - lac permease mutation allows uniform entry of IPTG into cells - allows to control induction level - "tighter control" 5) Origami - thioredoxin reductase, glutathione reductase mtuations to *enhance doulbe bond formation* 6) Mach1 = fast cell growth Actual protein production method from bacteria? - answer1) Vector of interest clone is grown at 37C in LB (lysogeny media - typrtone, yeast extract, NaCl,) overnight with ampicillin to select for bacterias not carrying plasmid - requries addition of fresh ampicillin 2) Grow to an *optical density of 600nm at 0.6* (exponential growth phase) 3) Grows mL to L of bacteria 4) Induce with IPTG 5) Spin down cells, lyse - get 1mg-500mg for every L of bacteria IPTG - answerIsopropyl-beta-D-thiogalactopyranoside A lactose "variant" that *cannot be metabolized* that will displace the lac reporessor from the lac operator (allowing T7 operator to be activated) IPTG induction is reversible - answerFalse; it is "permanent" on mode,b ecause it cannot be metabolized We induce (add IPTG) when... - answerWhen the OD at 600nm is approixmately 0.2 to 0.6 (bacteria is at its exponential growth phase) (This can be visualized) That is, get protein during the cell's maximal level of competance Lac operon is normally used by bacteria to... In the presnec of lactose, this operon is - answerExpress the beta-galactosidase, permease, and transacetylase proteins for lactose breakdown Lactose will bind to the *lac repressor* and inactivate it via inducing - allows transcription LacI - answerOn the lac operon will make lac inhibitor/repressor that binds to its own operator to repress transcription Bacteriophage T7 will bring... - answerInfects E. coli Its own RNA polymerase genes, so if it gets transcribed then the T7 RNA polymerase will be made and will bind to its own T7 promoter sequence