Genetic Engineering (GE-1) Lecture 2, October 2024, Nile University PDF

Genetic Engineering (GE-1) ABGEI-305 Dr. Mohamed Hazman Lecture 2 09-Oct-24 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 2 1 Methodology of Genetic Engineering Lecture 2 Dr. Mohamed Hazman 09-Oct-24 Dr....

Genetic Engineering (GE-1) ABGEI-305 Dr. Mohamed Hazman Lecture 2 09-Oct-24 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 2 1 Methodology of Genetic Engineering Lecture 2 Dr. Mohamed Hazman 09-Oct-24 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 2 2  General Guide Agricultural Production of a Recombinant Artificial desired product DNA modification of Industrial organism’s DNA Increase/decrease technology specific gene expression Medical 09-Oct-24 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 2 3  Genetic Engineering looks like… Chem. Bio Phys. Isolate and clone Recombinant DNA deliver to target tissue 09-Oct-24 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 2 4  II. The methodology of gene manipulation Host cells and vectors By Benchling 09-Oct-24 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 2 5  Getting DNA into the cell Plant genetic Bacterial Transfection Transduction Transformation Transformation Plants Bacteria or yeast Animal cells Prokaryotic or Agrobacterium or Chemical or high (typically) Eukaryotic (cell biolistic electric field Chemical (cationic line) bombardment Plasmid not lipid) or physical Viral injecting DNA Transient or chromosomal DNA (particle gun) Bacteriophage permanent Small size Transient or (bacteria), (inherited) (chemical), large permanent adenovirus (human) size plasmids No viral-agent (electroporation) 09-Oct-24 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 2 6  Host cell: getting workers back to the office Whatever how perfect cloning job you did, it is still in a test tube! Vectors carry the gene to host cell (living system) to permit propagating (molecular agriculture) Physical separation, The needle is made from the same material amplification and selection as the haystack 09-Oct-24 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 2 7  Types of commonly used host cells Monocots: Rice Dicot: Arabidopsis HUMAN!!!! 09-Oct-24 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 2 8  Somatic and germline human genome editing Gene-edited cells are already beginning to be used in clinical treatments for adults. genetically modifying embryos was – and is – far more ethically contentious..why? changes are made to every cell in the body passed down to subsequent generations Could be ever medically justified? Human embryo get an injection at a laboratory in China 09-Oct-24 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 2 9  Prokaryotic host: E.coli-the workhorse of genetic engineering Gram-negative bacteria Fully reproduce in 20 min Grow well in even low nutrition 4.6 x 106 bp Intense information more than any other organism on earth Fully sequenced (1/1000 human genome) Endless number of strains (ex: DH5α vs O157:H7) 09-Oct-24 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 2 10  Dethroning E.coli? Gram-negative Salt marches mud bacteria 5.2 x 106 bp Reproduce in the half time needed by E.coli, is that enough to replace E.coli? Scientists are skeptical as the research was published in bioRxiv Vibrio natriegens (not a peer-review platform) Time shall decide! 09-Oct-24 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 2 11  Successful cloning, but then to where? Gene clean E.coli Transformation 09-Oct-24 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 2 12  The right vector/host combination Isolate gene for structural analysis Expression in a certain organism (E.coli or yeast) (ex: plant cells) Shuttle vector Not necessarily mutually exclusive Stratagene, USA Takara, Japan 09-Oct-24 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 2 13  Find a way into E.coli: competency Chemical method (CaCl2) + heat shock Physical method: high electric field 1kV. (42 ℃) (reversible electroporation) 09-Oct-24 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 2 14  Troubleshooting: competent cells!! Pseudo-colonies: satellite or artifact Storage costs (-80 ℃) Consumes effort, money and time Fresh as long as it is frozen 09-Oct-24 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 2 15  The first step is the isolation of a target gene Cloning is moving genes around plasmids, wild or mutated genes Mining new genes (ex: RNAseq and GWAS) Genes with well-known function and sequence Genomic DNA library, and cDNA library Showed interesting function or expression pattern qPCR (RT-PCR): Reverse-transcriptase real time PCR PCR with another special primers (RE add- on sites) 09-Oct-24 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 2 16  Insert: a special PCR product Hazman, M. Gel express: a novel frugal method quantifies gene relative expression in conventional RT-PCR. Beni- Suef Univ J Basic Appl Sci 11, 11 (2022). https://doi.org/10.1186/s43088-022-00194-3 09-Oct-24 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 2 17  Vectors: Carrier 09-Oct-24 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 2 18  Vectors in the context of biotechnology DNA molecules used as tools to transfer a foreign DNA fragment of interest (FOI) to the host cell 1. Ensures that FOI or GOI (gene of interest) is propagated in host cell, otherwise will be degraded. cloning/propagation vector, ex: pUC19 and pBluescript II SK (+) 2. Ensures that FOI or GOI is stably expressed Image courtesy: Hazman et al., 2019 Expression vector, ex: pET-28 (+) 09-Oct-24 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 2 19  Plasmid: back-pocket genetic material of bacteria chromosome plasmid Small circular piece of autonomous replicating DNA linear or circular RNA plasmids exist (but are very rare) low-mid-high copy High copy number (small in size), low copy number (large in size as F-plasmid in E.coli) Must replicate otherwise get lost Hundreds of genes could be loaded on a single plasmid 50% of bacteria Cryptic plasmid: no phenotypic effect on host cell Can inhabit Yeast or fungi 09-Oct-24 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 2 20  Main three functions of plasmid in wild bacteria Sex factors: Small piece of DNA imparts bacterium’s ability to conjugate and transfer DNA to another recipient bacterium Colicin factors (anti-competitor agent) Bactericidal molecules (kill but do not lyse) other strains of the same family Resistance Transfer factor Transferring resistance traits to one or more drugs (ex: antibiotic) between the same family or different strains 09-Oct-24 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 2 21  Basic artificial plasmid (vector) components 09-Oct-24 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 2 22  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 09-Oct-24 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 2 23  Function of each basic element in plasmids 09-Oct-24 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 2 24  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 09-Oct-24 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 2 25  Types of Ori: A) High copy number, ex:pUC19 High copy number plasmid (700/cell) Under relaxed control Independent from host cell chromosomal replication 09-Oct-24 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 2 26  Types of Ori: B) Low copy number, ex: pSC101 REP101 Low copy number plasmid (20/cell) Under stringent control Replication is synchronized with host cell chromosomal DNA replication 09-Oct-24 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 2 27  Other factors control copy number/plasmid yield Insert toxic protein (dead cells or enforced low copy number) repetitive sequences (not stable Oops stage plasmid) ☺ Bacterial strain nonspecific endonucleases degrade plasmid (endA-E.coli might help) Growth conditions Lack of antibiotic, temp, nutrition, agitation, duration etc…. Culture inoculum Lack of fresh inoculum (do not use glycerol stock directly) 09-Oct-24 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 2 28  Thanks 09-Oct-24 Dr. Mohamed Hazman, School of Biotechnology, NU Lecture 2 29