1. Transformations of Cells (and Transfections too) Bacteria, Fungi, Plants, and Animal Cells & Recombinant DNA Technology

2. Transformation  Transformation (def): the genetic alteration of a cell resulting from the introduction, uptake and expression of foreign genetic material (DNA) in molecular biology  This can be done to Bacteria, Fungi, Plants, and Animal cells

3. Transformation - History  1928 - Frederick Griffith transforms nonpathogenic pneumococcus bacteria into a virulent variety by mixing them with heat-killed pathogenic bacteria.  Transformation was demonstrated in 1944 by Oswald Avery, Colin MacLeod, and Maclyn McCarty, who showed gene transfer in Streptococcus pneumoniae was pure DNA.  Avery, Macleod and McCarty call the uptake and incorporation of DNA by bacteria transformation.

4. Transformation - Mechanisms  Bacteria  transformation refers to a genetic change brought about by picking up naked strands of DNA and expressing it, and competence refers to the state of being able to take up DNA.  Two different forms of competence should be distinguished, natural and artificial.

5. Transformation - Mechanisms  Bacteria - Natural competence  Some bacteria (around 1% of all species) are naturally capable of taking up DNA. Such species carry sets of genes specifying machinery for bringing DNA across the cell's membrane or membranes.  The evolutionary function of these genes is controversial. Although most textbooks and researchers have assumed that cells take up DNA to acquire new versions of genes, a simpler explanation that fits most of the observations is that cells take up DNA mainly as a source of nucleotides, which can be used directly or broken down and used for other purposes

6. Transformation - Mechanisms  Bacteria - Artificial competence  Artificial competence is not encoded in the cell's genes.  It is induced by laboratory procedures in which cells are passively made permeable to DNA, using conditions that do not normally occur in nature.  These procedures are comparatively easy and simple, and are widely used to genetically engineer bacteria.  Artificially competent cells of standard bacterial strains may also be purchased frozen, ready to use.Common Strain of E. coli - DH5α (alpha)

7. Transformation - Mechanisms  Bacteria - Artificial competence - Temperature  Chilling cells in the presence of divalent cations such as Ca2+ (in CaCl 2 ) prepares the cell walls to become permeable to plasmid DNA.  Cells are incubated with the DNA and then briefly heat shocked (42 o C for 30-120 seconds), which causes the DNA to enter the cell.  This method works well for circular plasmid DNAs but not for linear molecules such as fragments of chromosomal DNA.  An excellent preparation of competent cells will give ~10 8 colonies per μg of plasmid. A poor preparation will be about 10 4 /μg or less. Good non-commercial preps should give 10 5 to 10 6 transformants per microgram of plasmid.

8. Transformation - Mechanisms What is a plasmid again?  A plasmid DNA molecule contains sequences allowing it to be replicated in the cell independently of the chromosome.  Plasmids used in experiments will usually also contain an antibiotic resistance gene which is placed in a bacterial strain that has no antibiotic resistance.  Therefore, only transformed bacteria will grow on a media containing the antibiotic.

9. Transformation - Mechanisms  Bacteria - Artificial competence – Electroporation  Electroporation is another way to make holes in cells, by briefly shocking them with an electric field of 100-200V/cm.  Now plasmid DNA can enter the cell through these holes.  Natural membrane-repair mechanisms will close these holes afterwards.

10. Transformation - Mechanisms  Bacteria - Artificial competence – Lipofection  Lipofection (or liposome transfection) is a technique used to inject genetic material into a cell by means of liposomes which are vesicles that can easily merge with the cell membrane since they are both made of a phospholipid bilayer.  The vescicle fuses with the cell membrane (similar to how two oil spots at the top of a broth will fuse) and the contents of the vesicle & the cell are combined.

11. Transformation vs. Transfection  Transfection (def): the introduction of foreign material into eukaryotic cells.  This typically involves opening transient pores or 'holes' in the cell plasma membrane, to allow uptake of material.

12. Transfection Mechanisms Yeasts and Fungi  These methods (and more) are currently known to transform yeasts:  Two-hybrid System Protocol:  The two-hybrid system involve the use of two different plasmids in a single yeast cell.  One plasmid contains a cloned gene or DNA sequence of interest while the other plasmid contains a library of genomic or cDNA. (later)  Frozen Yeast Protocol:  Frozen yeast cells that are competent for transformation after thawing.  Gene Gun Transformation:  Gold or tungsten nanoparticles can be shot at fungal cells growing on PDA, transforming them.  Protoplast Transformation:  Fungal spores can be turned into protoplasts which can then be soaked in DNA solution and transformed.

13. Transfection Mechanisms  Plants - A number of mechanisms are available to transfer DNA into an organism, these include:  Agrobacterium is a genus of Gram-negative bacteria that uses horizontal gene transfer to cause tumors in plants. Agrobacterium tumefaciens is the most commonly studied species in this genus. genusGram-negativebacteriahorizontal gene transfertumors Agrobacterium tumefaciens speciesgenusGram-negativebacteriahorizontal gene transfertumors Agrobacterium tumefaciens species  Horizontal gene transfer (HGT), also Lateral gene transfer (LGT), is any process in which an organism incorporates genetic material from another organism without being the offspring of that organism. By contrast, vertical transfer occurs when an organism receives genetic material from its ancestor, e.g. its parent or a species from which it evolved. Most thinking in genetics has focused upon vertical transfer, but there is a growing awareness that horizontal gene transfer is a highly significant phenomenon, and amongst single-celled organisms perhaps the dominant form of genetic transfer. Artificial horizontal gene transfer is a form of genetic engineering. offspringgeneticsgenetic engineeringoffspringgeneticsgenetic engineering

14. Transfection Mechanisms  Plants - A number of mechanisms are available to transfer DNA into an organism, these include:  Agrobacterium mediated transformation is the easiest and most simple plant transformation. Plant tissue (often leaves) are cut in small pieces, eg. 10x10mm, and soaked for 10 minutes in a fluid containing suspended agrobacterium. Some cells along the cut will be transformed by the bacterium, that inserts its DNA into the cell. Agrobacterium  Placed on selectable rooting and shooting media, the plants will regrow. Some plants species can be transformed just by dipping the flowers into suspension of Agrobacteria and then planting the seeds in a selective medium.  Unfortunately, many plants are not transformable by this method.

15. Transfection Mechanisms  Plants  Electroporation: make holes in cell walls using electricity, that allows DNA to enter. Electroporation  Viral transformation: Package your genetic material into a suitable plant virus and then use the modified virus for infection of the plant. Viral transformation Viral transformation  Genomes of most plant viruses consist of single stranded RNA which replicates in the cytoplasm of infected cell.  So this method is not a real transformation (why?) … since the inserted genes never reach the nucleus of the cell and do not integrate into the host genome.  The progeny of the infected plants is virus free and also free of the inserted gene

16. Transfection Mechanisms  Plants  Particle bombardment (gene gun): Coat small gold or tungsten particles with DNA and shoot them into young plant cells or plant embryos. Some genetic material will stay in the cells and transform them. This method also allows transformation of plant plastids.  The transformation efficiency is lower than in agrobacterial mediated transformation, but most plants can be transformed with this method.

17. Transfection Mechanisms  More on the “gene gun”  The target of a gene gun is often a callus of undifferentiated plant cells growing on gel medium in a petri dish. After the gold particles have impacted the dish, the gel and callus are largely disrupted. However, some cells were not obliterated in the impact, and have successfully enveloped a DNA coated tungsten particle, whose DNA eventually migrates to and integrates into a plant chromosome. calluspetri dishchromosomecalluspetri dishchromosome  Cells from the entire petri dish can be re-collected and selected for successful integration and expression of new DNA using modern biochemical techniques  Selected single cells from the callus can be treated with a series of plant hormones, such as auxins and gibberellins, and each may divide and differentiate into the organized, specialized, tissue cells of an entire plant. This capability of total re-generation is called totipotency. The new plant that originated from a successfully shot cell may have new genetic (heritable) traits. auxinsgibberellinstotipotencyauxinsgibberellinstotipotency

18. Transfection Mechanisms  Gene gun with Humans and Animals  Gene guns have also been used to deliver DNA vaccines to experimental animals. Theoretically, it may be used in humans as well. DNA vaccinesDNA vaccines  The delivery of plasmids into rat neurons through the use of a gene gun is also used as a pharmacological precursor in studying the effects of neurodegenerative diseases such as Alzheimer's Disease. Alzheimer's Disease Alzheimer's Disease  The Gene gun technique is also popularly used in Edible vaccine production technique, where the nano gold particles coated with plant gene under the high vacuum pressurized chamber is transformed into suitable plant tissues.

19. Transfection Mechanisms  Animals  Microinjection: use a thin needle and inject the DNA directly in the core of embryonic cells. Microinjection  Viral transformation: Package genetic material into a virus, which delivers the genetic material to target host cells. Viral transformation Viral transformation