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University of Waterloo
BIOL 342
Kyra Jones

Biol 342- Molecular Biotechnology Notes Chapter 1 Fundamentals of Molecular Biotechnology Biotechnology – use of organisms (their metabolism) to produce a product for a commercial use Molecular Biotechnology – DNA manipulation to enhance a natural product (protein/enzymes) e.g. insertion of human insulin into E.coli for insulin production Cohen, Chang, and Boyer‟s work  first demonstration of gene cloning using plasmids as vectors  however, thought to be dangerous if genes combined from two different organisms produced a novel organism with undesirable and dangerous properties Chapter 2 Molecular Biotechnology Biological Systems Prokaryotes Eukaryotes - Single chromosome (haploid) - Typically diploid - Circular chromosome - Linear chromosome - Contain plasmids - No plamids (except yeast) - No nucleus - Nucleus - Polycistronic mRNA (operons) - Transcription/translation - 1o transcript = mRNA (no introns) uncoupled - mRNA unmodified - Monocistronic mRNA - Transcription/translation - 1 transcript processed (introns) coupled - mRNA modified (5‟methylated - Initator tRNA carries N-f- cap and 3‟ methionine poly-A tail - Translation initiation at RBS - Initiator tRNA carries methionine (Shine-Dalgarno sequence) - Ribosome binds 5‟ cap, scans Chapter 3 DNA, RNA, and Protein Synthesis Prokaryotic DNA mRNA Protein Transcription Translation Eukaryotic DNA 1o transcript mRNA Protein Transcrip RNA 5‟cap, 3‟poly-A tail tion Proces Removal of introns sing Translation Gene: is a specific nucleotide sequence containing the elements required to produce RNA or protein ORF – contain start codon and gene. Does not contain stop codon Prokaryotic: DNA  5‟PO 4 P O RBS ATG TAG RBS ATG TAG T OH 3‟ Transcriptio n mRNA  5‟ SD AUG UAG RBS AUG UAG OH 3‟ PO4 5’ UTR RB 3’ UTR S Translation Shine-Dalgarno Sequence in E. Coli = *AGGAGG* Protein  NH – n-f-Met (first aa) –aa-aa-aa-aa-COOH 2 Stop Codons = UAG, UGA, (last amino acid) UAA Note: 2 proteins produced in this case Operon: cluster of genes that are regulated and under the control of an operator e.g. lacZ operon  operator repressed, no expression unless inducer present Eukaryotic: DNA  5‟PO 4 P Exon1 Intron1 Exon2 Intron2 Exon3Intron 3 T OH 3‟ Transcript ion o 1 transcript  5‟G cap Exon1 Intron1 Exon2 Intron2 Exon3 Intron3 AAAAAA OH 3‟ Post Transcriptional Modification mRNA  5‟G cap Exon1 Exon2 Exon3 AAAAA OH 3‟ Translati on Protein  NH –Met (first aa) –aa-aa-aa-aa-COOH (last amino acid) 2 mRNA processing in Eukaryotes – Post transcriptional modification 1) 5‟Cap – loading signal for ribosomes  therefore Euk cannot translate polycistronic mRNA 2) 3‟poly-A tail  stability and protects mRNa  Assists in translation 3) Slicing of introns/exons Chapter 4 Recombinant DNA Technology Recombinant DNA cloning procedure Source with Insert Circular Cloning Vector Enzymatic Enzymatically fragmentation linearize Insert Vector DNA Clone insert into Vector DNA (circular) Transform into Host cell Isolate cells with cloned genes Produce protein from cloned gene Restriction Endonucleases (Type II) – specific way to cut DNA - Cleaves double-stranded DNA at a specifically recognized sequence (palindromic)  reads same forward and backward in 5‟to 3‟direction - Used by bacteria in restriction modification and protection against foreign DNA - Produces „sticky‟/ „cohesive‟ends OR blunt ends Sonication - Non-specific, random, produces variety of blunt-ended Glass beads fragments - Hard to control and reproduce (size of frags. and location of cuts) Use DNA Ligase to catalyze phosphodiester bond formation – blunt ends harder to ligate due to no base pairings to hold ends together Restriction Fragment Length Polymorphism (RFLP) 1) Used to identify genetic differences in closely related strains of microorganisms or segments of DNA - Gives you guide on how closely related strains are 2) Digest genome with a specific combination of RENs and look for differences in the resulting gel pattern 3) Used to trace movement of pathogen strains through a population Presence of a different band on the gel may indicate: 1) Change in nucleotide sequence (substitution) 2) There has been an insertion 3) There has been a deletion Result of these: REN sites may have been created or removed Plasmid Cloning Vectors - Self replicating, double-stranded, circular DNA molecules maintained in bacteria as extrachromosomal entities - Range in size from <1 kb to > 500kb - Have varying copy numbers  controlled by genes on plasmid and host interactions - Segregation  some plasmids encode information to ensure they are distributed to new daughter cells  ensures they are perpetuated (or else lost) 5 Common Features 1) Origin of Replication - must be functional in host cell to be used - Some vectors engineered with more than 1 Ori for use in diff. species (Shuttle Vectors) 2) Selectable Markers - When vector or vector+insert is present  we can see it phenotypically - To differentiate from other cells not carrying plasmid/insert 3) Multiple Cloning Sites - Several unique REN sites for cloning = more choices 4) High Copy # - So lots can be made 5) Small Size - Allow for larger inserts - Maximize transfer into host pBR322 - 2 Antibiotic resistance genes (selectable markers) Tetr and Ampr - OriC for E. Coli - Small size 4300 - High copy # 50 copies/cell - Unique REN sites Recombinant plasmid =construct = Insert + Vector - If you insert into PstI site then you get disruption of the Ampr Gene and the cells will not grow on Amp media so you know There is insert Problems with Single Digests 1) Vector self-religation 2) Vector-Vector ligation (Vector Concatamers) 3) Insert-Insert ligation (Insert Concatamers) 4) No control in the orientation of the insert To avoid these problems, you can do 1 of the following: 1) Alkaline Phosphatase treatment of vector DNA (pg 59) - Enzymes that remove phosphate groups from DS DNA molecules (do the treatment on the plasmid, so you have – OH on 5’and 3’ends on both strands of plasmid) - Ligase cannot join –OH and –OH groups (prevents vector religation and vector concatamers) - Only colonies that result will be those of vector +insert - The OH-OH nick between plasmid and insert will be fixed inside cell when plasmid replicates 2) Do a Double Digest - Avoids vector self-ligation, and insert concatamers (vector concatamers can still occur) - Uni-directional cloning EcoRI in PBR322 what if you use this?? - You will need to make colonies because you have both the resistance markers - You can run them on a gel and determine the size since you know the original size and the cut will produce a linear plasmid so the bigger will have the insert in there and the smaller are just the inserts alone - You can also use PCR These are used when you can't select Transformation – introducing free exogenous DNA into a bacterium or yeast cell - Not efficient, most don’t survive, must select those that carry the construct - Competence can be increased by heat-shocking cells or electroporation creating transient openings in cell wall that enable DNA molecules to ender the cytoplasm Selectable Markers Using pBR322 – Screening = 2 Step Process Selectable Markers = ampicillin and tetracycline …if insert was inserted into amp gene and transformed into E. Coli…..then: 1) Plate E.Coli cells on media containing tetracycline/incubate overnight - Those that grow have vector 2) Select and number some of the resulting individual colonies and transfer onto media containing ampicillin 3) Those that don’t grow have a non-functional ampicillin resistance gene  have insert 4) Look back at tetracycline media and locate colonies with the vector +insert What if you needed to use the EcoRI recognition site of pBR322? How would you distinguish those carrying the desired construct from those carrying relegated vector-only? 1) Grow on tetracycline or ampicillin media - If insert in amp r gene, then grow on tetracycline and vice versa - Those that don’t grow don’t have vector 2) Select colonies 3) Restriction digest then run Gel Electrophoresis - One band = Vector that self-ligated - Two bands = Vector + Insert pUC19 Advantages 1) MCS - More options due to more REN sites and adjacent to one another - Prevents alkaline phosphatase step  avoids self-ligation if 2 diff. REN sites used as well as promotes uni- directional cloning imp for mapping and expression of gene product - All in one place so you don’t have overlaps (won’t disable both selectable markers) 2) Color selection + antibiotic selection (1 step) 3) Very small plasmid (smaller than pBR322) 2600bp - Therefore can hold bigger inserts 4) Very high copy #100 copies/cell lacZ’ – lacZ part of E.coli lac operon ( codes for β-galactosidase) - Deconstruct lacZ polypeptide into 2 subfragments o C-terminal lacZω(host encodes) & N-terminal lacZα (plasmid encodes) o Need to come together to get functional enzyme/colour o Together = β-galactosidase that converts X-Gal from colorless to blue in Presence of inducer IPTG (lacZ normally repressed) O Therefore, use a media containing Amp, X-Gal, and IPTG - lacI – encodes repressor protein – binds to operator in absence of IPTG o Prevents expression of lacZ’ MCS is in-frame with lacZ’ within the 5’end therefore a functional β-galactosidase subunit is produced when no insert is present. Therefore colonies that lack insert appear blue. Presence of insert in MCS would not produce the β- galactosidase subunit. Therefore colonies with insert would appear colorless. Host Cell Features 1) Have recombination systems disabled so foreign DNA won’t integrate with host chromosome as well as prevent recombination among plasmids 2) Have REN system disabled Advantages using E.Coli Disadvantages using E.Coli 1) Well characterized with many 1) Not good at expressing defined strains proteins from foreign sources 2) Highly competent (gram +ve and euk) 3) Grows quickly - Lack of recognition of 4) Safe to work with promoter and RBS - Lack of posttranslational processing ability (glycosylation) Genomic Libraries - A collection of cloned DNA fragments that together contain the entire genome of the organism of interest Uses - Genome sequencing - Isolating a particular DNA fragment or gene of interest Construction 1) Cut DNA into overlapping fragments 2) Insert all fragments into vector 3) Transform constructs into host cells (=library) 4) Identify and isolate colonies carrying fragment of interest Construction of a Genomic Library Do a partial digest with REN that cuts relatively frequently to generate lots of overlapping fragments Partial digestion by: 1) Shorten incubation time 2) Decreasing amount of REN 3) Decreasing temperature As duration treatment extended (15), cleavage occurs at an increased number of sites How can you determine if a genomic library is “complete”? N= ln (1-P) / ln (1-f) N = number of clones Completely P = probability of finding a specific gene digested f = average insert size (bp) / size of entire genome (bp) Partially However: 1) Some fragments are too large to clone (REN sites are far digested apart) 2) Difficult to clone highly repetitive sequences (polymerase slips or palindromic sequences) To solve: Create more than one library using different combinations of RENs Eukaryotic Gene Libraries (cDNA libraries) Make cDNA libraries instead because you have no need to clone bunch of introns unless for sequencing Reverse transcribe mRNA cDNA Considerations of cDNA libraries: 1) The cDNA molecules cloned are only specific to a specific tissue (expressed in one tissue but not another) under specific conditions (time/temp) 2) Gene expression in Eukaryotes is tissue/condition specific! - Need basic background information regarding protein product (like where it might be found) before constructing a cDNA library Construction of a cDNA Library Method 1 1) Isolate mRNA - Exploit the 3’poly-A tail in Euk. mRNA by column elution using oligo dT magnetic beads Oligo dT is bound to magnetic bead so it only hybridizes to polyA tail. Column elution removes rest of RNA 2) Convert mRNA to cDNA – First strand synthesis - Oligo dT act as primer for Reverse Transcriptase (RT) - Catalyze synthesis of DNA strand using RNA as template Problem: mRNA tends to form secondary structures (base-pair with itself) therefore difficult for RT to scan and replicate  results in truncated DNA strands 3) Convert mRNA to cDNA – Second strand synthesis - RT forms hairpin loop at the end, forming a 3’ OH end - RNAse H (works on Hybrids) treatment to degrade RNA component - Hairpin has exposed 3’OH used as starting point for Klenow (fragment of DNA pol. I  no proofreading ability so synthesis is fast) and add in dNTPs to form DS DNA 4) Remove Hairpin Loop - Hairpin loop is single-stranded  degraded by S1 nuclease - Elute from magnetic beads - Can now be cloned by blunt-end ligation (S1 produces blunt-ends) or add DNA linkers to containing REN recognition sites to the ends Disadvantages with Method 1: 1) Blunt-end ligation difficult (prefer double digests to produce cohesive ends) 2) Produces incomplete/truncated DNA fragments Method 2: Modificatio ns: 1) Addition of trehalose  allows reaction to occur at high temperature  stabilizes RT o Prevents self-hybridization of mRNA 2) Oligo-dT has built-in REN recognition sites (primer adaptor) at 5’ end for cloning REN Site 3) Incorporate methylated dCTP into first-strand means treatment with REN for cloning will not cut cDNA molecule o Your fragment may have REN sites that you don‟t know about & might be cut when you do double digest followed by ligation to vector 4) Biotinylation o Biotin attached to ends of mRNA molecule o Treatment with RNase I (digests ss RNA) leaves full-length RNA/DNA molecules present for isolation by biotin-streptavidin binding o Those that contain biotin at the ends would bind to streptavidin and therefore be isolated, others are washed off; only full length DNA, prevents truncated DNA Full Length Removed Trunca ted Removed RNase H treatment 5) RNase H treatment 6) Add poly (dG) tail to 3‟end of the first free strand cDNA 7) Add poly (dC) primer adaptor to hybridize with poly (dG) tail o Used for second-strand synthesis and later cloning o DNA ligase added for any nicks during synthesis o RNase H to prevent RNA from hybridizing cDNA vs genomic libraries Eukaryotes - cDNA library when searching for structural genes - not interested in cloning introns or intergenic sequences - genomic for sequencing or analyzing regulatory elements ( P, O) Prokaryotes - genomic (not cDNA since absence of polyA tail) - Pro. Genome is small, therefore cloned genes code directly for product Vector Choices Bacteriophage λ ~ 50 kb linear DS DNA, host = E. coliK12 Lytic – infects host, makes lots of virus, lyses and release of viral particles Lysogenic – infects host, genome integrates into host chromosome, virus particles not formed For use in cloning vector, do NOT want lysogenic cycle, therefore we replace the genes that control lysogenic integration  make a replacement vector 1) Remove middle ~20kb Replace with 2) Clone in DNA of ~20kb insert between the two arms and it will get replicated and packaged into viral particles ~50 kb for Advantage of viral vectors  high efficient infectivity Very efficient delivery of DNA vs transformation packaging DNA replication by rolling circle mechanism - Viral endonuclease cleaves at each cos site and packaged into head - Same amount of DNA packed in each virus Add empty heads and tails and recombinant phage genome and the resulting recombinant viral particles can be used to infect E.coli (transfection) Zones of lysis (plaques) If insert size = too small  then the phage capsid head would not fold properly If insert size = too big  then the DNA would not fit into the head Cosmids Transfected like a bacteriophage Propagated just like a plasmid Features: ~ 45 kb Contains E.coli Ori Contains tetracycline resistance gene - Cos sites anneal and circularizes DNA when transfected into E. coli - Results in tetracycline- resistant colonies carrying cosmids as plasmids - Highly infectious Artificial Chromosomes – carry huge amounts of DNA (100 to 2000kb) Bacterial Artificial Chromosome (BAC) - Big plasmid can hold big amounts of DNA once non-essential genes are removed - Stably maintained o Holds > 300 kb of insert Yeast Artificial Chromosome (YAC) - Designed to mimic yeast chromosome o Linear, telomeres, ori, centromere o Holds > 800 kb of insert Human Artificial Chromosome (HAC) - Developed for propagation in human cultured cells o Holds > 2000 kb of insert Vector system Insert Capacity (kb) Plasmid 0.1 – 10 Bacteriophage λ 10 – 20 Cosmid 35 - 45 Bacteriophage P1 80 - 100 BAC 50 – 300 YAC 100 - 2000 HAC >2000 Screening methods for libraries 1) By DNA hybridization - Use a labeled DNA or RNA probe (complementary sequence) - Match between probe and target DNA must be at least >80% and over a segment of at least 50 NTs long - Useful if a gene in another organism is similar to the human gene, can use the gene of another organism as probe (conserved genes) - Disadvantage: gene may not be 100% identical due to genetic code degeneracy i) By autoradiography - Probes are labeled with radionucleotides 32 P-dCTP - Hybridization results in radioactive signal detected using radiographic film - Results in black spots on a clear background film = autoradiograph 2 Major Disadvantages - Safety  exposure to radiation unsafe for worker - Radiodecay  weakening of signal over time, therefore only effective for a short period ii) By fluorography (chemiluminescent detection) - DIG labeling = not radioactive - Gives off light when activated - Safe and can be kept for long periods of time, label is stable - Results in black spots on clear background on film = Fluorograph Chemiluminescent substrate acted upon by alkaline phosphatase and gives off light iii) Chromogenic detection (colour) Chromogenic substrate acted upon by alkaline phosphatase - Results in blue spots on nitrocellulose membrane 3) By looking for production of the protein i) Immunological screening  Inject into animal and obtain serum full of antibodies  Use when no probe available and don’t know the sequence of the gene of interest  Note: not all hosts can produce the proteins encoded by inserts  Therefore host must be able to use promoter and RBS elements from the cloned DNA and have posttranslational modification system ii) Screening by protein activity  Colonies exposed to a substrate for which there is a phenotypic result = differential media  For this method, one must know characteristics of protein (what its substrate is, is it inside the cell or exported) in order to use the proper media iii) Screening by functional complementation  Use host cells defective for the particular gene of interest  Use minimal medium (have only one carbon source) = selective media  Cells that carry the functional copy of the inserted DNA will grow CHAPTER 4 CHEMICAL SYNTHESIS, SEQUENCING, AND AMPLIFICATION OF DNA Nucleotide = Phosphate + base + sugar - Deoxy = missing OH in 2’position - OH 3’position - Phosphate 5’position Nucleoside = Base + sugar Chemical structure of a single strand of DNA 3’OH from one NT + 5’ Phosphate of another  forms a phosphodiester bond - This is what happens in cells  requires cellular enzymes Automated Chemical Synthesis of DNA – Phosphoramidite method - Glass bead act as solid support, chains synthesized on this - Spacer molecule added to capture next base from solution like a “fishing line” - Removed after completion of synthesis Synthesis is in reverse: 3’to 5’!Adding to 5’end! 1) Add first nucleoside, 3’OH attached to spacer o Contains DMT group on 5’carbon which act as a “cap” Second nucleoside contains DMT cap AND 3’ phosphite/diisopropylamine group = phosphoramidite nucleoside 2) Remove 5’DMT by acid hydrolysis (TCA) leaves 5’OH for addition 3) Add tetrazole to activate phosphoramidite so it can form phosphite triester bond 3’phosphate to 5’OH 4) Add acetic anhydride and DMAP  adds cap to all 5’OH groups that have unreacted (cap after every addition of phosphoramidite)-> acetylation 5) Oxidize phosphite triester bond to form stable pentavalent phosphate triester bond (Me group still there) 6) Do Modifications o Acid hydrolysis to replace DMT with phosphate and remove spacer o Final purification to separate full length strands from truncated strands o Remove methyl group from phosphate triester bond Coupling efficiency - For every cycle, not all growing chains will receive a phosphoramidite - Therefore results in fewer and fewer full length chains being formed at each cycle - There is a limit to the length of DNA chain that can be made (~100NTs) - The longer the target chain is, the smaller # of the chains formed will be full length Synthetic Genes To create longer DNA  you synthesize each strand separately as to have overlap at the ends of each fragment Anneal these fragments, and have DNA polymerase fill in the gaps by utilizing the 3’OH groups of each strand. DNA ligase seals the nicks. Sequence this synthetic gene to ensure it is correct. Uses of synthetic Genes and why we want to synthesize DNA molecules? 1) Can produce a protein product without having the actual gene  sequence protein and design a gene to code for this protein even though it may not be the same as the authentic gene. 2) To design primers or probes, linkers and adapters PCR method much faster - Conceived by Dr. Kary Mullis - Uses heat stable Taq polymerase, DNA template, 2 flanking primers, dNTPs - Denature (95C), anneal (50-70C), synthesis (75C) Adv: - Each cycle doubles the amount of DNA - Very small samples needed - Easily detected Linkers - Short double stranded, blunt-ended DNA molecules that contain a REN site - They are blunt-end ligated to blunt ended DNA molecules (ie cDNA) Adapters - Variants of linkers which can introduce new REN sites into vectors for cloning Primers for PCR Usually short, 16-45 bp in length Design a 22-mer primer set containing an EcoRI site (GAATTC) and PstI site (CTGCAG) for cloning of the ORF Denaturation and synthesis More synthesis by PCR Sequence PCR products to ensure you have the right sequence!! By Sanger’s method of sequencing (chain termination) or automated sequencing. Pyrosequencing - Fast and cheap but gives less nucleotide reads compared to classic cycle sequencing - ~300 bases vs 1000 bases respectively - Useful for high-throughput projects  sequencing projects 1) Denature Template 2) Add primer (at end of vector, just before beginning of insert) 3) Add polymerase, APS, ATP sulfurylase, luciferase, luciferin, apyrase, computerized addition of 1 random nucleotide in series of dCTP, dATP, dTTP, dGTP Since it’s no match, (CTP not incorporated by polymerase) therefore degraded by apyrase dCTP dCMP, polymerase cant use 4) Add next random nucleotide eg. dATP Although apyrase stays in vessel, can limit it from degrading NT that needs to be added to the sequence by modifying the ratio of concentration of NT & apyrase 5) Before next NT is added, remove existing ATP and free NTs from reaction by apyrase. No need for a washing step!! 6) Keep adding dNTPs and record when you get a signal Primer walking 1) Design primer to known sequence 2) Sequence to however far you can 3) Use the information from the first read to design a second primer for the next walk 4) Walk back along opposite strand to confirm sequence Problem: Suppose you have isolated and sequenced a protein from a bacterium but you do not know the gene that encodes this protein. How would you find it? 1) Screen a library of another organism that has a similar protein 2) Inject protein in rabbit to obtain serum with lots of antibodies (immunological screening) 3) Design synthetic DNA probe based on protein sequence Designing Synthetic Probes based on protein sequence – ‘Rev erse T ra n slation ’ Make probe based on amino acid sequence of protein Problem – genetic code is degenerate Solution – will have to make enough probes that cover all the possible combinations of codons involved Exceptions to the genetic code: Genetic code isn’tquite that universal Codon Usage/Frequency Different organisms prefer to use some codons over others for the same amino acid = codon bias 2 = 4  synthesizing 4 probes would have high probability of detecting the gene from library screening rd Usually only 3 position of a codon is different for the same a.a  leads to redundancy Chapter 6 - Manipulation of Gene Expression in Prokaryotes Why would we want to do this? - to maximize the yield of foreign (recombinant) protein - GOAL -E. coli is often, but not always the bacterium of choice -fast generation time -grows to high density -simple nutritional requirements -grows under a variety of temperatures (4C - 42C; 37C optimal) -genetics, molecular biology, physiology, biochemistry of E. coli are THE most studied to date -variety of mutants available as hosts for cloning -variety of specialized vectors exist -transforms with high efficiency Transcriptional control in pro- Promoters - The strength of the promoter is imp to have strong binding to sigma factor - Sigma factor is imp for the intiation of transcription by providing the increased binding of the holoenzyme to the DNA at the promoter - RNA polymerase holoenzyme = sigma factor + core enzyme (4 subunits) - Sigma factors specifically recognize and bind the promoter regions to initiate trans. once the trans. is initiated the SF is released and trans. begins Promoter is characterized by two regions -10 (TAATA) and -35 (TTG) - These regions are highly conserved - Differ between species because of the differences in the SF - There are altnernate SF recognize specific promoters on highly regulated genes and/or those genes expressed under particular conditions - Some genes have multiple promoters used by various sigma factors for regulation under specific conditions - Strong’ promoters match the consensus perfectly - ultimate binding with principle sigma factor - the more deviation from the consensus, the ‘weaker’ the promoter - distance between the -10 and -35 consensus is also important for initiation - sequence between these sites can also ‘fine tune’ binding Transcriptional control of E.coli Negative trans. control - Binding of repressor to operator to stop trans. - Lac operon is OFF-> ON - it has a negative control where the repressor lacI is on the operator and it will stop transcription but when the inducer IPTG is there then it will change the conformation (allosteric effect) of the repressor and make it not functional  transcription occur. Another example of –ve trans. control - Trp transcription is usually ON when the trp is not present since it is a corepressor that have allosteric effect on the repressor to make it active and bind the DNA and the repression is working transcription is off +ve trans. control - The positive control of trp is when the activator bind the activator binding site (upstream of promoter, not operator) and enhance the transcription that induce the conformational change in the DNA to facilitate binding by RNA polymerase holo-enzyme. - The effector will bind the activator and inactivate it to make the level of the transcription normal again. Why regulate the promoter protein? Why not have it constitutively? o The protein could be toxic to heterologous species o Take lot of energy and efforts which will slow down the division of the cell o Cells can halt essential metabolic system and die or cease to divide o Large quantities of benign protein can have toxic effect o Want to prevent the recombinant until we have large densities of the cells Expression vectors - Vectors used to max recombinant protein production - Many of the E.coli expression vectors use lac and trp operons promoters - Usually use a hybrid promoter Tac promoter is a hybrid promoter - lac (-10) and trp (-35) operons - -10 region has altered nt sequence that makes it stronger than the WT -10lacUV5 created from exposure to UV light - Spacer region of 16 bp Trc promoter - Spacer region of 17 bp Adv. Of trc and Tac  Both trc and tac promoters are stronger than trp (3x) and lac (10x) promoters  They match closely the consensus sequence for the E.coli principle SF  Modifications in the spacer region made them more efficient than the WT they were derived from Disav. Of trc and tac - Sometimes these vectors are leaky  trans. not 100% off - Maybe not enough LacI repressor so the repression should be modified - These plasmids are designed with the lacI gene added to increase the level of repression to minimize leakiness o Some E.coli strains have mutations lacIq that have higher level of repressor o Also can take adv. Of catabolite repression  IPTG and cAMP levels have to be high to get the max transcription level  cAMP is imp in the transcription of the lac operon where it binds catabolite activator protein CAP +ve trans. control and increase the affinity of the RNA polymerase to bind the promoter.  Glucose is a catabolite repressor (indirect) where it reduces the level of cAMP present  Lactose will be broken down to glucose and galactose so we don’t want it in the media when we want to increase the level of transcription of beta galactosidase. o We add IPTG since it is inducer analog to lactose Conditions to get the max expression of lac gene? -grow up cells in presence of glucose to maximize catabolite repression and do not add lactose/IPTG - minimizes transcription -to induce high levels of transcription, move the cells to a medium without glucose and lactose (use an alternative carbon source such as mannose or fructose). Lactose breaks down into glucose and galactose, so we don’t want it there. -and add IPTG (analog of lactose) to induce (derepress) -produces maximum level of transcription The pL promoter of bacteriophage λ - controlled by a repressor protein (encoded by cI) at operator OL - mutation in gene cI produces a temperature-sensitive repressor protein which is inactive at 42C - can include these genetic elements in a plasmid or cosmid in order to tightly regulate transcriptional control - grow host cells at a lower temperature of 28C to 30C at which the repressor is fully functional Transcription prevented. -when culture reaches desired stage of growth, switch to 42C. Repressor inactivated transcription proceeds. Plasmid pCP3 - E.coli expression vector - P (strong) and O (-ve control) by temp sensitive cI repressor (active at low temp) - MCS downstream of the P/O to insert the target gene (including RBS) - Ori highly active at 42C where the repressor is not active r - Selectable marker Amp Plasmid pCP3 -grow cells carrying the construct at 28C - repressor is active, no transcription - copy number is low due to mut
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