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Microsoft powerpoint - mbb 343- ch06- l07 & 08- 091702-091902-- handout draft- with deletions.ppt

Topics for lecture 4 (090502)/ lecture 5 (091002):Chapter 4:I.
Intro. to bacterial plasmids as cloning vectors Digestion (a.k.a. restriction or cleavage) of DNA withrestriction endonucleases– a.k.a. restriction enzymes Ligation of pieces (a.k.a. fragments) of DNA together Cloning pieces of DNA using bacterial plasmids Putting plasmid DNA into bacteria cells-- transformation Topics for lecture 6 (091202):Chapter 5:I.
III. DNA amplification by polymerase chain reaction (PCR) Reminder: Take-home Quiz will be distributed this Thurs, Sept. 12and is due at beginning of class on Tues, Sept. 17.
Sequencing of DNA, chemical synthesis of DNA, and amplification of DNA bypolymerase chain reaction have revolutionized the way molecular biologyexperiments are done. Illustrated by the following Nobel awards: -- 1980 Nobel Prize in ChemistryThe prize was divided, one half being awarded to: PAUL BERG for his fundamental studies of the biochemistry of nucleic acids,with particular regard to recombinant-DNA and the other half jointly to: WALTER GILBERT and FREDERICK SANGER for their contributionsconcerning the determination of base sequences in nucleic acids.
-- 1993 Nobel Prize in ChemistryThe prize was awarded for contributions to the developments of methodswithin DNA-based chemistry equally between: KARY B. MULLIS for invention of polymerase chain reaction (PCR) method. and MICHAEL SMITH for fundamental contributions to the establishment ofoligonucleiotide-based, site-directed mutagenesis and its development forprotein studies.
A. Done by machines using computer operated valves 1) amino groups of bases adenine, guanine & cytosine are derivatized by addition of benzoyl, isobutyryl & benzoyl respectively, to preventundesirable side reactions but, thymine is not derivatized b/c it has no amino group 2. DNA is synthesized by solid phase synthesis-- DNA strand is attached to a a. allows all reactions to take place in one tube b. after each step, unreacted chemical is washed away 1) Starting complex for chemical synthesis of DNA oligonucleotide-- Fig. 5.2 2) Phosphoramidite-- have these for all four nucleotides A, C, G, T– Fig. 5.3 3) Remove dimethoxytrityl (DMT) group from nucleotide linked to spacer and bead– Fig. 5.4 4) Capping- must inactivate unreacted nucleosides– Fig. 5.6 5) Add the next phosphoramidite & linking reaction occurs 6) Then must stabilize the phosphite triester formed– Fig. 5.7 8) Cleave oligonucleotides from the spacer 10) Phosphorylate 5' end of each oligo.
Efficiency must be >98% at each step in order to get goodyield of complete oligonucleotide– Table 5.1 Yield of complete oligonucleotide also depends on length Topics for lecture 7 (091702)/ lecture 8 (091902): I. Vectors for expression of proteins in bacteriaII. Approaches to improve "transgene" expression A. Fusion protein productionB. Tandem gene arraysC. Increasing translationD. Increasing protein stabilityE. Overcoming oxygen limitationF. Insert transgene into chromosomeG. Secrete protein III. Integration of foreign genes in host chromosomesIV. The biological cost of "transgene" expression I. Vectors for expression of proteins in bacteria A. General1) Insertion of a gene into a plasmid & transformation into a host do NOTassure expression of the encoded protein 3) Lots of experimentation may need to be done to optimize expression B. Features of "the system" altered to modulate expression: C. Establishing strong and regulatable promoters Regulation allows for precise control of expression insert random DNA fragments in front of a reporter gene and look
for increased expression-- Fig. 6.1
D. Examples of promoters that have been developed: 1) lac promoter-- promoter from lac (lactose metabolism) operon of E. coli 2) trp promoter-- promoter from Trp (Trp metabolism) operon of E. coli 3) tac promoter-- the -10 region of lac promoter PLUS the 4) pL promoter-- promoter from bacteriophage lambda 5) T7 promoter-- promoter from bacteriophage T7 1) Regulation by the lac promoter-- Fig. 6.2: a) presence of lactose (or a simulated lactose molecule such as IPTG) results in repressor protein off operator c) binding to promoter is increased by cyclic AMP (cAMP) which is at a higher conc. when glucose is low b) If Trp conc. made low (or by addition of 3-indoleacrylic acid) get derepression (so gene turned "on") 3) pL promoter-- controlled by cI repressor of bacteriophage or by temperature sensitive mutant of cI called cI : b) so the gene for T7 pol. is inserted into the E. coli genome under the control of the E. coli lac promoter c) Add IPTG and get increase production of T7 pol. by derepressing the lac promoter, which then transcribes the transgene behind the T7 promoter d) one key the “tightness” of this system is the repressor to promoter ratio Increasing protein production in E. coli cells Plasmid pPLc2833 was created for max. protein production in E. coli cells expression from pPLc2833 was increased by replacing its origin of replicationwith that from plasmid pKN402, which increases copy number 5- to 10- fold,making plasmid pCP3-- Fig. 6.3 & Table 6.1.
It is not explained in book exactly why raising the temp. to 42 oC increasesnumber of plasmids– presumably expression of some gene involved in copynumber controlled by the temp. sensitive cI promoter is increased in expression Two plasmid system to regulate transgene expression- Fig. 6.4: Used for large-scale production of proteins in E. coli b/c easy to manipulate: -- when want transgene “off”– just grow in molasses plus casein hydrolysate (very abundant protein casein “cut-up” with proteases– enzymes that cut-up other proteins) -- when want transgene “on”– add “tryptone” (protein called peptone “cut-up” with protease called trypsin), which.
Transgene expression in other bacteria– Fig 6.2: Various promoters were tried in various bacteria 2) tac most active in E. coli, least active in other bacteria Plasmid pAV10 has promoter regions that make it useful for expression in many types of bacteria– Fig. 6.5. II. Approaches to improve "transgene" expression Solution– “attach” the cloned protein to a stable host protein i.e. make a chimeric, fusion protein The “fusion” is based on making the appropriate construct at the DNA level (as you did for Question 4 of Quiz 1) All that is required is that you know the sequence of both genes and put them together in a way that maintains the reading frame so that the protein encoded by the cloned gene is expressed properly May have to cut apart (cleave) fusion protein b/c: 1) whole protein may not be suitable for clinical use Solution: add a protease cleavage site between protein regions-- Fig. 6.6 Usually leaves a non-Met end, but this is usually OK-- see stability later 1) used to make antibodies to the protein encoded by the cloned gene One example is putting the cloned gene behind the ompF coding region- Fig. 6.7 Different fusion protein "partners" bind to different "ligands“ that can be usedfor purification-- Table 6.3 Purification of fusion proteins containing the "Flag" peptide attached-- Fig. 6.8.
Often proteins, when expressed in E. coli cells, end up in insoluble form in"inclusion bodies"-- just a name for the area in the cells where proteins get stuck If couple proteins to thioredoxin, proteins remain soluble even when fusionprotein made at high level-- Fig. 6.9 4) Way to screen for proteins of low abundance in a library II. Approaches to improve "transgene" expression Level of gene expression in part depends on number copies of the transgene So, ligate fragments containing transgene into a vector in such a way thatmultiple copies are inserted-- tandem arrays To ensure all in same orientation, use AvaI, which is a RE with anon-palindromic site Three factors greatly influence translation efficiency: 3) Lack of secondary structure formation in the 5’UTR of the transcript-- orcould prevent ribosome complex from binding OR stop it from moving-- Fig. 6.13 Example of a vector designed for optimal transcription & translation-- pKK233-2 (Fig. 6.14) 4) ATG (of non-transcribed strand = “coding strand”) is 8 nt 5) transcription terminators from bacteriophage lambda II. Approaches to improve "transgene" expression Half-life (a fancy way to say how long something stays around) of proteins varyfrom hours (slow turnover) to minutes (fast turnover).
Obviously, if you want to over-express a protein, slow turnover is better.
1) extent of disulfide bond formation-- more is better but how many should -- true in both prokaryotes & eukaryotes Regions rich in P, E, S, & T-- especially when surrounded by clusters of(+) charged amino acids are more susceptible to proteolytic degradation(cleavage by proteases).


Summary The present thesis investigates the transmission history of Sörla saga sterka , one of the younger fornaldarsögur . The methodological background to the study is the sociology of texts and material philology. The aim of the study is threefold: First, to trace the transmission history of the saga; second, to examine possible connections between the socio-economic background of sc


Clinical and Experimental Allergy, 37, 166–173Continued need of appropriate betalactam-derived skin test reagents for themanagement of allergy to betalactamsM. Blanca , A. Romano , M. J. Torres , P. Demolyz and A. DeWeck‰ÃAllergy Service, Carlos Haya Hospital, M ´alaga, Spain, Department of Internal Medicine and Geriatrics, UCSC-Allergy Unit, Complesso Integrato Columbus, Romeand IRCCS

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