Lab Report 2: PGLO TRANSFORMATION EXPERIMENT
Doris Daniels
PURPOSE: The purpose is to transform E. coli bacteria by adding plasmids that allow the bacteria to glow green under UV light in the presence of arabinose sugar. Also, it is to observe if bacteria will grow in the presence of ampicillin and without ampicillin using two –DNA plates and two +DNA plates.
INTRODUCTION: In this lab experiment, we will perform genetic transformation by putting some new DNA into E. coli cells. When observing bacteria it frequently has one or more small circular pieces of DNA called plasmid and one large chromosome. The plasmid DNA normally has genes for more than one trait. Researchers utilize a process called genetic engineering to inject genes coding for
…show more content…
Therefore, management happens at an exact particular location on the DNA template, called a promoter, here RNA polymerase suppress the DNA and starts transcription of the gene. Inside bacteria, varieties of connected genes are constantly conglomerated and transcribed into RNA from the individual promoter. The indicated conglomerates of genes reserved by an individual promoter are called operons. The three genes (ara B, araA, and araD) will code the three digestive enzymes included in the operation of arabinose being a conglomerate in what called the arabinose operon. The indicated three proteins are subordinate on the start of transcription against an individual promoter that is reserved by a DNA protein named araC. The DNA code of the pGLO comprehension is designed to involve visible feature of the arabinose operon. Together the promoter and the araC gene are existing. Still, the genes, that code the arabinose breakdown, araB, A, and D, provide substitute by the individual gene that codes for the Green Fluorescent Protein (GFP). As a result, in the company of arabinose, araC proteins encourage the joining of RNA polymerase and GFP is created. Cells glow a very bright green color as they create additional protein. If arabinose is not present araC will not assist in the progress of the binding of RNA polymerase and the GFP gene is no longer transcribed. If the GFP …show more content…
Then a procedure known as heat shock will need to be carried out.
In order for transformation cells to develop in the presence of ampicillin, they will need nutrients, and a little time to incubate so their new acquired genes can be expressed.
It is suggested that the Ca2+ cation of the transformation solution (50 mM CaCI2, ph 6.1) counteract the offensive negative charges of the phosphate determination of the DNA and the phospholipids of the plasma membrane, grant the DNA to get into the cells.
The heat shock boosts the absorbency of the cell membrane to DNA. Although the method is unknown, the event of the heat shock is important and has been made perfect for the kind of bacteria used and changing conditions take on.
The ten minutes process of development to take place followed by the extra LB nutrient broth permits the cells to develop and demonstrate the ampicillin resistance protein beta-lactamase. in order for transformed cells to continue to live on the following ampicillin selection plates. The retrieval culture could be incubated at 37 degrees overnight to boost the transformation adeptness above
The first step of the experiment was ligation, and the objective was to insert EGFP cDNA into a restriction cut pET41a(+) vector to obtain a recombinant plasmid that would express green fluorescent gene. pET41a(+) was the choice of vector to ligate the EGFP into. Its structural design and genomic sequential properties render it especially well-suited for cloning and high-level expression of peptide sequences. This 5933 bp circular vector contains a built in sequence for Kanamayacin resistance gene. “Rooting of non-transgenic shoots was completely inhibited in all culture media containing kanamycin” (Montserrat, et. al., 2001). This allowed the growth of recombinant and non-recombinant colonies of E. coli, all of which contained the vector insert.
While the previous experiment identified colonies containing recombinant DNA, the patching experiment distinguished which colonies contained the hlyA fragment and which ones did not. Colonies that could cause haemolysis of the blood agar plate indicated that recombinant DNA taken up contained the hlyA fragment ligated with pBluescript, which is the desired subcloning product. The hlyA fragment contains the hlyA gene which encodes for a haemolytic protein that causes the red blood cells in the blood agar to lyse. Therefore, non-haemolytic colonies were transformed with pBluescript plasmid ligated with the pK184 fragment and were not able to cause haemolysis as no hlyA gene was present. In theory, this experiment allowed for the aim to be achieved as it identified colonies with the desired product. Inoculating certain colonies in broth culture allowed for gel electrophoresis to be carried out and confirm if the aim of the experiment has been
The ligation was expected to make four combinations. The original pBK-CMV and CIH-1 fragments would region to make a non-recombinant pBK-CMV/CIH-1 plasmid. The original pUC19 fragments would rejoin to make a non-recombinant pUC19 plasmid. The larger fragment of pBK-CMV and the small 27bp fragment of pUC19 or the desired recombinant vector, CIH-1 fragment and the larger 2659bp pUC19 fragment. As pBK-CMV does not contain the ampicillin gene then transformed Ecoli containing these would not to survive on the Agar leaving only pUC19 recombinants and non-recombinants.
Another weakness being not effectively utilizing the heat shock method of genetic transformation, the transformation solution of calcium chloride could not have gotten cold enough when sitting on the ice bath for certain periods of time. This lowers the effectiveness of heat shock genetic transformation as the plasma membrane of the cells do not become permeable enough to be able to take up the foreign DNA that it is being exposed
The expression of lac operon in each tube equals the amount of beta-galactosidase produced. Therefore, by looking at the amount of beta-galactosidase under different conditions collectively is a good way to understand the function of inducers and repressors in supervising the expression of lac operon and the control of gene expression generally.
Transformation of T87 cells was done by culturing the cells in B5 medium supplemented with 1 μM 1-naphthaleneacetic acid (NAA) and 40 g L-1 sucrose. The cells were cultured for one day at 22°C with continuous illumination and shaking at 120g. Next, 10 μL of overnight cultured Agrobacterium transformed with respective vectors were added into the cell suspension and cultured for an additional two days. After co-cultivation, the cell suspension was washed thrice with 10 mL of JPL3 medium supplemented with Carbencilin (250 μg mL-1) by centrifuging at 100g for two minutes. Finally, the cells were resuspended and spread onto JPL3 selection agar plate supplemented with Carbencilin (250 μg mL-1), Kanamycin (30 μg mL-1) an...
It has an outer membrane that contains lipopolysaccharides, a periplasmic space with a peptidoglycan layer, and an inner cytoplasmic membrane. It also consists of adhesive fimbriae. Some strains of E. coli are piliated and are capable of accepting, as well as transferring plasmid to and from other bacteria. This enables the bacteria under stressful or bad conditions to survive. Although its structure is simple with only one chromosomal DNA and a plasmid, it can perform complicated metabolism to help maintain its cell division and cell growth. E. coli produce very rapidly; a single microscopic cell can divide to form a visible colony with millions of cells overnight (phschool.com). It is the preferred bacteria in most laboratories because it grows fast and easy, and can obtain energy from a wide variety of sources. Since the birth of molecular cloning, E. coli has been used as a host for introduced DNA sequences (biotechlearn.org.nz). In 1973, Boyer and Cohen showed that two short pieces of DNA could be cut and pasted together, and returned to
The operon is a set of coding regions of DNA clustered together that includes structural genes and it is under the control of a single regulatory region. The operator regulates transcription, which is a repressor protein. When the operator binds to a segment of the regulatory region, transcription is shut down.
The two modes of analysis that will be used to identify an unknown insert piece of DNA would be plating the transformation cells onto LA plates that have either ampicillin or chloramphenicol and PCR. We will use the PCR thermocycler to denature the restriction enzymes that were specifically used to assimilate the vector DNA. It is important to use the PCR thermocycler because denaturation of the restriction enzyme will prevent the restriction enzyme from cutting the vector DNA, after the insert DNA has assimilated to the vector DNA. After the addition of specific primers that complement the base pair to its corresponding target strand, PCR will be used. Subsequently, Taq polymerase will be used to determine whether the insert DNA has been properly assimilated to the vector DNA. Within this specific situation, the target strand will be the insert DNA. After we let the PCR thermocycler run for approximately 2 ½ hours, we will then put our PCR products in the gel and run the gel to completion. After the gel has run to completion, we will then take a photograph of the gel using the UV transilluminator with the assistance of our TA. If the insert DNA was properly assimilated to the vector DNA, then our corresponding gel photo would have one band. After the cells have been transformed, we would g...
In bacteria, RNA polymerase attaches right to the DNA of the promoter. You can see how this process works, and how it can be regulated by transcription factors, in the lac operon and trp operon videos.
Structural genes code for the enzymes themselves. RNA polymerase transcribes all of the genes into a polycistronic mRNA. The promoter is the site where the RNA polymerase binds to the DNA prior to beginning transcription. The operator serves as the binding site for the protein called the repressor. The regulatory gene encodes the repressor protein.
The birth of genetic engineering and recombinant DNA began in Stanford University, in the year 1970 (Hein). Biochemistry and medicine researchers were pursuing separate research pathways, yet these pathways converged to form what is now known as biotechnology (Hein). The biochemistry department was, at the time, focusing on an animal virus, and found a method of slicing DNA so cleanly that it would reform and go on to infect other cells. (Hein) The medical department focused on bacteria and developed a microscopic molecular messenger, that could not only carry a foreign “blueprint”, or message, but could also get the bacteria to read and copy the information. (Hein) One concept is needed to understand what happened at Stanford: how a bacterial “factory” turns “on” or “off”. (Hein) When a cell is dividing or producing a protein, it uses promoters (“on switches”) to start the process and terminators (“off switches”) to stop the process. (Hein) To form proteins, promoters and terminators are used to tell where the protein begins and where it ends. (Hein) In 1972 Herbert Boyer, a biochemist, provided Stanford with a bacterial enzyme called Eco R1. (Hein) This enzyme is used by bacteria to defend themselves against bacteriophages, or bacterial viruses. (Hein) The biochemistry department used this enzyme as a “molecular scalpel”, to cut a monkey virus called SV40. (Hein) What the Stanford researchers observed was that, when they did this, the virus reformed at the cleaved site in a circular manner. It later went on to infect other cells as if nothing had happened. (Hein) This proved that EcoR1 could cut the bonding sites on two different DNA strands, which could be combined using the “sticky ends” at the sites. (Hein). The contribution towards genetic engineering from the biochemistry department was the observations of EcoR1’s cleavage of
Distinct characteristics are not only an end result of the DNA sequence but also of the cell’s internal system of expression orchestrated by different proteins and RNAs present at a given time. DNA encodes for many possible characteristics, but different types of RNA aided by specialized proteins sometimes with external signals express the needed genes. Control of gene expression is of vital importance for an eukaryote’s survival such as the ability of switching genes on/off in accordance with the changes in the environment (Campbell and Reece, 2008). Of a cell’s entire genome, only 15% will be expressed, and in multicellular organisms the genes active will vary according to their specialization. (Fletcher, Ivor & Winter, 2007).
Secondly the gene has to be cut from its DNA chain. Controlling this process are many restriction endonucleases (restriction enzymes). Each of these enzymes cut DNA at a different base sequence called a recognition sequence. The recognition sequence is 6 base pairs long. The restriction enzymes PstI cuts DNA horizontally and vertically to produce sticky ends.
The duration of the experiment should be increased as the thermal death times of B. subtilis at 60, 70 and 80°C were unable to be determined within 110 minutes. The duration can be increased to 180 minutes so as to better investigate its thermal death times. If the presence of bacterial growth was still observed after 180 minutes of exposure, it can be assumed that B. subtilis is able to survive well in that temperature. An exposure time of one day can be carried out to confirm this assumption.