Genetic Engineering (GE1) Lecture 3 PDF

Genetic Engineering (GE-1) Dr. Mohamed Hazman Lecture 3 18-Oct-24 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 3 1  Basic elements of plasmids ABGEI-305 (Lecture 3)...

Genetic Engineering (GE-1) Dr. Mohamed Hazman Lecture 3 18-Oct-24 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 3 1  Basic elements of plasmids ABGEI-305 (Lecture 3) Dr. Mohamed Hazman 18-Oct-24 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 3 2 1. The bacterium Vibrio natriegens is suggested to be an alternative to E.coli because of: a. Its tolerance to high concentrations of salts b. Ultrafast growth rate c. It could be propagated in labs using lower cost growth media d. It is not pathogenic to humans 2. The initiation of DNA replication depends on proteins that recognize and bind to a DNA sequence motif in case of: a. Bacterial chromosomal DNA b. Theta type A plasmids. c. Low copy number plasmids b. Any of the above 3. ………………………….. is the transfer of DNA into cells through viral agent. a. Induction b. Transduction c. Infection d. Transfection 4. The plasmid pUC19 is a high copy number plasmid that is used regularly in protein expression (T ) (F) 5.Gene edited crops have higher WTC and WTP values than crops produced by recombinant DNA technology (T) (F) 18-Oct-24 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 3 3 The discovery of microRNA wins the 2024 physiology Nobel Prize Ruvkuns Ambros 18-Oct-24 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 3 4  Reading vectors: basic elements Origin of replication (Ori) Multiple Cloning Site (MCS) Selectable Marker Promoter Sequence elements of translation Essential sequence elements in the backbone of a vector 18-Oct-24 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 3 5  Function of each basic element in plasmids 18-Oct-24 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 3 6  1. Origin of replication (Ori) in the host cell Ori ensures that the vector can self- replicate in the host cell using [its] replication machinery Controls copy number per cell Initiating replication by DNA polymerases enzymes and others High A-T and low G-C content, why? Replicon consists of Ori and other supplementary sequences 18-Oct-24 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 3 7  Types of Ori: 1) Low copy number, ex: pSC101 Low copy number plasmid (20/cell) Under stringent control Replication is synchronized with host cell chromosomal DNA replication 18-Oct-24 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 3 8  Types of Ori: 2) High copy number, ex:pUC19 High copy number plasmid (700/cell) Under relaxed control Independent from host cell chromosomal replication 18-Oct-24 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 3 9 Chromosomal DNA replication in prokaryotes 18-Oct-24 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 3 10  Theta plasmids! The most dominant type of plasmid in enterobacteria (gram-negative, rode-shaped, flagella containing, and lactose fermenting ) Ex: Escherichia, Shigella, Salmonella, Yersinia. Well-studied due to ecological and clinical relevance coordinated leading- and lagging-strand replication, where lagging-strand replication is discontinuous. This leads to the generation of bubbles in the early stages of replication that resemble the Greek letter θ (theta) under the electron microscope Tight plasmid copy number regulation is essential because of the major and direct impact plasmid gene dosage has on gene expression on the plasmid and the host. Two types of host-replication mechanisms: DnaA dependent → Theta type A PriA-dependent pathway →Theta type B, C and D Theta type B adopts ColE1-like plasmid Theta type C adopts ColE2-like plasmid Theta type D adopts ColE3-like plasmid 18-Oct-24 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 3 11  Theta plasmids! 18-Oct-24 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 3 12  Theta type A plasmids: Striking Balance Low copy number plasmid: pSC101 Copy number is always limited by the use of the host cell's initiation proteins Replication initiation protein: Chromosomal DNA : DnaA Stringent plasmids: DnaA (coded by chromosomal DNA and Rep (coded by plasmid itself) Rep: Replication-encoded initiation Protein Handcuffing (two plasmids and Rep) bacterial chromosome Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 3 13  Theta type B plasmids: relaxed but for a reason ColicinE1 (ColE1) producing plasmids of E.coli Used to terminate (kill but not lyse= bactericidal) other types of bacteria by: o Inserted in target bacteria PM o Leaking nutrients and proton influx High copy number (concentration) in each single cell Originally (in wild type) max 40 copies per cell More related to Gram negative bacteria as E.coli 18-Oct-24 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 3 14  Artificial improvement in ColE1 wild plasmid Colicin gene is removed The ori was kept Antibiotic resistance gene was added as ampR or bla (beta- lactamase gene) Theta plasmid contains ColE1- derived origin of replication (ori) 18-Oct-24 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 3 15  Theta type B plasmid PriA-dependent pathway: resembles replication restart after following replication fork arrest→ extra DNA strand in needed Protein regulation is not required, just RNA primer of app 600 nt transcript (RNAII) which is encoded by Ori Anti-sense RNA (RNAI) molecules negatively regulate the replication process pMB1 ori (20 copy), pUC19 (700) ori in pUC19 differs only in two mutations compared to pMB1 The mutations impacted: Increased promoter activity Primer less sensitive to inhibition by RNA I Stabilize secondary structure for primer formation 18-Oct-24 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 3 16  Theta type B plasmid (independent from host cell replication) R-loop R-loop formation is the only essential step, if DNA polymerase III RNAseH and Pol I are missed, initiation still extending both strands possible, albeit at reduced efficiency. 18-Oct-24 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 3 17  Relaxed vs Stringent plasmids Relaxed plasmid (Type B) Stringent plasmid (Type A) High copy number per cell (ex:700) Low copy number per cell (ex: 20) Gene propagation/expression Likely with toxic protein genes Less restricted to host cell DNA replication More restricted to host cell DNA replication machinery machinery Multiple copies at any time of growth When bacterial undergo cell division PriA-dependent assembly DnaA-dependent assembly Replication is partially independent from host cell Replication is synchronized with host cell replication chromosomal DNA RNA molecules synthesized by the plasmid Requires enough amount of REP101 protein itself (no protein is needed) for replication Ex: pBluescript II SK Ex: pSC101 18-Oct-24 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 3 18  Thanks 18-Oct-24 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 3 19