The Human Of Recombinant Bacteria

134). They are loops of DNA that are separate from the chromosomal DNA and can self-replicate in a cell, found mostly in bacteria (Brown, 2011; Addgene, 2015). Lederberg and William Hayes discovered that plasmids were being transferred from one cell to another, not the chromosomal DNA (Brown, 2011, p. 135). This discovery lead to plasmids being an essential tool for scientists. Scientists can engineer plasmids to have specific genes to introduce into new cells (Brown, 2011, p. 134). On a plasmid loop there will be an origin of replication (ORI) and a multiple cloning site (MCS) where the gene of interest is inserted (Bio-Rad, 2015). This region has specific restriction enzyme recognition sites, which are cut by the enzymes to open up the DNA where the new gene will be inserted (Jove Science Education Database, 2015). Most plasmids will also contain an antibiotic resistance gene allowing cell survival in environments containing antibiotics (Jove Science Education Database, 2015).

Lab Report on pGAL Transformation In order to understand this lab the student first needs to understand how recombinant DNA is formed. To begin, the student extracts a plasmid, which is a circular strand of DNA found within bacterial cells, from the bacteria. Restriction enzymes begin to cut the plasmid at certain sequences of nitrogenous bases.

Our research on recombinant DNA mainly consisted of two experiments: Transformation and gel electrophoresis. In our first experiment, four microfuge tubes were given to us: pKAN DNA, pAMP DNA, unknown DNA, and a TE buffer without DNA. The two positive controls, pKAN and pAMP, consisted of an antibiotic resistance gene respectively to their name. The pKAN plasmid contained the gene resistance for kanamycin while pAMP carried the gene resistance for ampicillin. The negative control, TE, only contained buffer without DNA. The fourth tube was our unknown plasmid, which was either pKAN or pAMP; and by way of artificial transformation, we would be able to initiate the identification of our unknown plasmid.

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].

In this case, the DNA is the only thing left of the organism, so the process of cutting it from the source does not necessarily need to be done. In the laboratory scientists will use the process of restriction enzymes to cut the DNA into smaller parts. The cuts are made at specific nucleotide sequences. Different restriction enzymes recognize and cut different sequences of DNA. Restriction enzymes work by matching up with a space that matches the restriction enzyme. The site that it matches up with is called the recognition site. The restriction enzyme wraps around the DNA, and it causes a break in both strands of the DNA molecule. Restriction enzymes are often used in the process of DNA cloning. (Biotechnology Learning Hub,

the bacterial chromosome is genetically engineered and the plasmid is used to help the bacterium replicate.

In order to manipulate, clone and express certain genes it is essential that a greater understanding of how the gene in question operates, in order to isolate and potentially recreate that particular characteristic in a host organism (Strand ect al. 2003) Escherichia coli is almost the most used organisms of preference in lab based experumentation for the manufacturing of recombinant proteins. Its used as a mobile factory because it is stable and it has become the most famous expression platform organism to use. (Lee ect al.

As mentioned before, restriction enzymes, which can isolate DNA fragments, are needed for the DNA cloning process to occur. Restriction enzymes can be found in bacteria; these enzymes can but within the molecule, so they are called restriction endonucleases. Restriction enzymes in prokaryotes can selectively cut foreign DNA in the restriction process; during the process, the host DNA will be protected by a methylase, which is a modification enzyme that modifies the DNA and prevents cleavage. These two processes combined are called the restriction modification system.

Conjugation is another mechanism through which bacteria transfer resistance genes among each other. Conjugation uses mobile genetic elements (MGEs) such as plasmids and transposons as vehicles to share valuable genetic information (Manson et al., 2010). Lastly, the use of integrons allows bacteria to accumulate multiple resistance genes on their chromosomes (Jose et al., 2015). Integrons are site-specific recombination systems that recruit open reading frames in the form of mobile gene cassettes. These mobile gene cassettes are then incorporated into the host chromosomes, resulting in the addition of new genes along with appropriate machinery to ensure that the new genes are expressed adequately in the host cell (Jose et al., 2015; Thomas et al., 2005).

The use of recombinant DNA technology has been a huge advancement to science. In the early 1970s the first recombinant DNA was produced (“1972 First”, 2013). Researchers Stanley Cohen and Hebert Boyer used restriction enzymes to cut DNA at specific sites, and then fused them back together to form their recombinant vector (“1972 First”, 2013; Cederbaum, S., et al., 1984). Through transformation the vector is then inserted into a host cell to amplify their hybrid DNA, also known as recombinant DNA (“1972 First”, 2013). This technology allowed scientists to insert a human gene into the genetic material of common bacteria. This was a huge break through for science and medicine. Now the recombinant DNA could be used to produce protein encoded by the human gene (“1972 First”, 2013; Cederbaum, S., et al., 1984).

The infection by undocumented DNA starts the acquisition of viral DNA. Upon the detection of the invasion of bacteriophages, bacteria defend themselves in a timely fashion by inserting bits of viral DNA, the protospacer, into their chromosome at the end of CRISPR locus. To maintain the structure of CRISPR array, bacteria initiate the replication of a repetitive DNA sequence, the repeat.

The physical work of genetic engineering couldn’t have been done without the tools of biotechnology i.e. enzymes that cut he DNA molecules in order to select the wanted/ remove the superfluous genes. These specific enzymes came to be known as restriction enzymes. In nature it needs to be able to recognize and cut very specific factions of foreign DNA therefore protecting the bacteria against the unknown substance in a process called restriction. The bacterial cell then continues to protect its own DNA from restriction by adding –CH3 to adenines or cytosines within making the strand recognizable by the restriction enzyme as to avoid being cut. In biotechnology EcoRI acts as our restriction enzyme and makes a cut into the sugar phosphate backbone of the DNA in a section it can recognize. The complementary

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

Alcanivorax borkumensis is a bacteria that found in aquatic areas of the world. It is an unusual bacteria because it has the ability to break down oil found in the oceans. In the following pages, I will discuss the size and shape of the bacteria. Also, I will discuss how the bacteria’s environment and its unusual ability may help mankind in controlling pollution of the world’s oceans.

Plasmid DNA with Restriction Digest: The purpose of restriction digest of plasmid DNA is to understand how each DNA plasmids was cut with the given restriction enzymes and perform gel electrophoresis to observe the samples. Nine restriction digests were created, containing three digests for each of the three plasmid DNAs identifying as recombinant, non-recombinant, and unknown. Out of the nine digests, six are actual digests and three are undigested controls. A master mix is created to add to each of the nine samples with its following stock ingredients: 10 ul of 2X Reaction Buffer, 1 ul of Nco1, X ul of sterile water (Single digest), 10 ul of 2X Reaction Buffer, 10 ul plasmid DNA, 1 ul Nco1, 1 ul of Not1, and X ul of sterile water (Double