Biology: Bacterial Transformations

This lab is about moving genes from one thing to another using plasmids. Plasmid has the ability to replicate, so it replicates independently, and separately from the chromosomal DNA. Plasmid are one or more small piece of DNA and they enter cells as a double strand DNA. When they enter the cell as a doubke strand they do not invade he chromosomal DNA. We will also transform bacteria into GFP which is mainly from the jelly fish Aequorea Victoria. The GFP causes the the jelly fish to fluorescent and glow in the dark. After the transformation, bacteria starts to make the GFP which causes them to glow a green color under a ultraviolet light.

The purpose of this lab is to use genetic engineering to transform E. coli bacteria by inserting the plasmid pGLO, and to then see if the bacteria was transformed by using the antibiotic, ampicillin.

Transformation is the transfers of virulence from one cell to another, through the transferring of genetic material. It was originally postulated in 1928 through the works of Federick Griffith, a British microbiologist. Griffith observed that the mutant form, non-virulent form, of the bacteria Streptococcus Pnumoniae could be transformed into the normal, virulent form, when injected into mice along with heat killed normal forms. He concluded that somehow the information the dead virulent form had transformed the mutant form into a virulent form.

For this experiment, E. coli was best for genetic engineering because of their size, and their fast reproduction (Spilios, 2017). E. coli will be genetically transformed using an engineered plasmid. A plasmid is a circular piece of DNA which independently replicates and multiplies because it has its own origin of replication (Spilios, 2017). The pGLO is the plasmid used in this experiment. Plasmids are used as vectors and they contain manipulated genes such as genes coding for antibiotic resistance for drugs like ampicillin. This antibiotic resistance of such serves as the selectable marker in genetic transformation and for genetic transformation to proceed, the cell must reach competency which is the physiological state that is required for the vector plasmid to get into the cell for transformation (Spilios, 2017). While competency can be reached naturally in some organism, it must be reached artificially in E. coli through treatment with CaCl2 and exposing them to heat shock using incubation (Spilios, 2017).

The enzyme also cuts above and below the gene of interest and then ligase bonds the plasmid to the gene of interest and you now have a strand of recombinant DNA. Such a strand is useful because scientists can import useful genes into bacteria and these can be used to create such substances such as insulin. This makes life much easier for diabetics who cannot produce sufficient quantities on their own. On the opposing view, people may not accept this way of producing proteins as natural due to the fact that the DNA was genetically engineered. Eventually recombinant DNA will become high in demand because our bodies will adapt to their supply of proteins.

Certain E. Coli strains are also known to show resistance to bacteria killing antibiotics. This resistance is due to the plasmids, or small round DNA molecules, in the bacteria that carry the resistant genes. R Plasmids (resistance plasmids) are widely studied and bestow resistance to factors that inhibit growth of the organism. R plasmids code for proteins that can

Tiffany O’Connor                                                         November 9th, 2017              Plasmid Transformation      Purpose: Plasmid transformation is the process of transferring foreign exogenous DNA into a host cell to change its phenotype.   Theory and Background: Plasma transformation can occur naturally when a cell alters its genetics by taking in DNA from its environment. Genetic transformation can also be induced in a laboratory setting, called artificial transformation. Artificial transformation is accomplished by choosing a competent bacterium and introducing a plasmid.   E. Coli is the bacteria used in this lab.

One of the four ways that bacteria can increase their genetic diversity is by conjugation. This is a process by which a bacterium can transfer genetic material to another bacterium of different mating type, through direct contact. During conjugation one bacterium is going to be the receptor of the genetic material while the other is going to be the donor. The donor bacterium is going to grow a tube-like structure called a pilus, which is going to be used to contact the other bacterium and to transfer the genetic material. Usually these genetic material is going to have the form of plasmid, which are small circular pieces of non-chromosomal DNA. Another method that bacteria have developed in order to acquire different DNA is transformation. These is a process by which some bacteria can get pieces of DNA from the external environment, under certain circumstances. Transformation occur naturally in Bacillus, and some Streptococcus and Staphylococcus species. The third means by which bacteria obtain new DNA is by the acquisition of plasmids. These plasmids can travel between bacteria of the same or different species, and the act like parasitic pieces of DNA that infect bacteria. Usually these plasmids are not required for the normal function of bacteria and is often expel by them, unless it contains material beneficial

Plasmids are small double stranded circular non chromosomal DNA molecules containing their own origin of replication. Hence, they are capable of replication independent of the chromosomal DNA in bacteria. Plasmids present in one or more copies per cell, can carry extra chromosomal DNA from one cell to another cell and serve as tools to clone and manipulate genes. Plasmids used exclusively for this purpose are known as vectors. The genes of interest can be inserted into these vector plasmids creating a recombinant plasmid. Recombinant plasmids can play a significant role in gene therapy, DNA vaccination, and drug delivery [Rapley, 2000].

Bacterial transformation is the process of moving genes from a living thing to another with the help of a plasmid.The plasmid is able to help replicate the chromosomes by themselves; laboratories use these to aid in gene multiplication. Bacterial transformation is relevant in everyday lives due to the fact that almost all plasmids carry a bacterial origin of replication and an antibiotic resistance gene(“Addgene: Protocol - How to Do a Bacterial

My best bacterial friend has genes that has antibiotic resistance that I must have. There are many different ways for me to get ahold of them or transferred into my genes. One way to receive the gene is by the use of a plasmid. A plasmid is circular piece of replicating dna that is separate from chromosomal dna. In order to manipulate the plasmids, i will need to mutate them by using restriction enzymes, PCR, and ligation. Being able to use restriction enzymes to cut portions of genomes and have them inserted into a plasmid, make it easy for mutations (mutagenesis) to occur. Also, PCR can enable mutagenesis. Each plasmid is mapped out with information that includes the restriction enzyme sites and the origins of their replications. I could

Plasmid DNA is an autonomously replicating circular DNA molecule that is only about 1,000-20,000 base pairs in size and is separate from bacterial chromosomes. Plasmids are a significant part of molecular biology because they are small enough to be cloned or modified, they endure self-replication, they are stable over long periods of time and they have a diverse range of gene expression. Having these characteristics allows plasmids to introduce genes into bacteria. The difference between genomic and plasmid DNA is that genomic DNA is DNA that is part of an organisms chromosomes, while plasmid DNA is a small loop which is separate from the organisms chromosomes.

A suicide system in E.coli plasmid vector is used to reduce the potential risk of gene escaping into the environment. Genetic transfer from GM bacteria to other bacteria via trans-conjugation will cause unexpected genetic combinations. This method is implemented by using recipient plasmid with suicide gene gef from E.coli, with expression controlled by Pm promoter. Pm promoter sequence, which present upstream of the gef suicide gene will control the expression of gef suicide gene.

Recombinant bacteria is a bacterium cell that has had its genome changed, by the alteration, or edition of a gene. Recombinant DNA is DNA that has been artificially prepared by cutting DNA into smaller fragments and joining fragments together, that would not normally be found together naturally- even fragments taken from different organisms.( Principles of Gene Manipulation sixth edition, S.B. Primrose, R.M. Twyman and R.W. Old ) DNA can be "cut" using the enzyme Restriction endonuclease, which recognises a specific sequence of base pairs- called recognition sequences (Genetics Fourth Edition- Hugh Fletcher, Ivor Hickey). These enzymes are secreted naturally by bacteria as a defence mechanism. The enzyme cuts between the bases within this sequence in a "staggered pattern", leaving what are known as "sticky ends", which are off branches of single-stranded DNA (http://www.rpi.edu/dept/chem-eng/Biotech-Environ/Projects00/rdna/rdna.html). A vector, such as a plasmid in the case of a bacterium, is then cut with the same restriction enzyme, DNA polymerase is then used to add nucleotide bases by complementary base pairing to the sticky ends from two different cut strands. DNA ligase can then join these two strands into a single strand of rDNA (http://www.ncbi.nlm.nih.gov/books/NBK21881/). This vector, usually a plasmid can then be taken up by bacteria cells (transformation) becoming part of their genome. After it is taken up by the host cell, the plasmid is able to replicate, which

A gene must be transferred from one plasmid to another in order to give that organism a selective advantage e.g. antibiotic resistance. However, Bacteria have been industrialized that are resistant to antibiotics such as penicillin which is a result of the misuse of antibiotics. Resistance arises as changes or mutations within the bacteria’s genes develop, which help bacteria grow that are resistant to antibiotics and therefore have an advantage. From researching journals, I found that Pseudomonas Aeruginosa resistance poses threats to human and animal health as the body no longer has the defences to protect against the bacteria. Altogether 77 isolates were tested and out of 77, 13 where negative for the presence of class 2 integrase gene (Olga et al, 2016). Only the most beneficial bacteria are selected to carry plasmids and so natural section is an important factor so that only those of the greatest advantage are selected. There are various different types of plasmids some of which include F-plasmids for expression of the sex pilli, resistant plasmid that provides resistance against antibiotics or poisons, col plasmid which contain proteins that kill or