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
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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
Once the recombinant plasmid was obtained, it was then inserted into E. coli cells through transformation. From a successful transformation, we expected the bacterial cells to translate the inserted EGFP sequence into its protein form. The bacteria cultures were plated on petri dishes containing growth supplement, Luria Broth (LB), an antibiotic: Kanamycin, and IPTG which induced the fluorescence property within successfully transformed bacterial colonies. Different variants of the petri dishes were also included as control and unknown.
Figure 2 shows the results of the electrophoresis. Lanes 5 and 7 indicate the fragments obtained when the plasmids are digested with both restriction enzymes, indicating the approximate fragment size for the hlyA gene, the pK184 plasmid and the pBluescript plasmid. This is useful for identifying the recombinant DNA needed for this experiment
...et light. If the LAA plate glows green under exposure to ultraviolet light, then we can conclude that our unknown insert piece of DNA would be the kan gene. If it does not glow green under exposure to ultraviolet light, then then we streak the colony from our LAA plate onto the LAC plate using a sterile glass spreader. When the LAC plate is dray, we place it upside down in the microfuge rack so that it can be incubated at 37 ºC. Incubation at 37 ºC will allow the transformed bacterial cells to grow. If we see bacterial growth on the LA plate containing chloramphenicol, we can conclude that our unknown insert piece of DNA would be the cat gene, since the cat gene is resistant to chloramphenicol. Afterwards, we then grab the microfuge tube labeled NP and repeat the aforementioned steps shown above pertaining to the LA plates. This would be considered our control.
pBK-CMV is a plasmid vector 4518 in size, it also contains a multiple coding site (polylinker) that has recognition sequences for many restriction endonucleases. cDNA molecule CHI-1, which is 600bp, has been previously inserted. pUC19 is a cloning vector developed by….. in …….at….(REF). This vector is 2686bp in size and contains a 54 base pair (bp) polylinker containing 13 specific restriction sites, Xba1 and EcoR1 inclusive. It makes a good cloning vector as it is small in size, this makes it easier to be taken up by its host during transformation and allows for a faster replication time (Green, 2015). It contains an origin of replication pMB1 which is essential to be able to replicate. pMB1 has a high copy number allowing for multiple copies to be made (REF hcn pmb1). The pUC19 plasmid vector contains an ampicillin resistance gene, the host containing this plasmid will survive in the presence of ampicillin allowing for the selection of transformed host bacteria. The polylinker of pUC19 is contained within a lacz’ gene allowing us to distinguish between recombinant pUC19 and non-recombinant pUC19 through a process call insertional inactivation (Green, 2015).
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.
In this experiment the heat shock method will be used to deliver a vector (plasmid) of GFP to transform and grow E. coli bacteria. Four plates containing Luria Bertani (LB) broth and either –pGLO and +pGLO will have E. coli bacteria added to it. The plate containing –pGLO (no pGLO) and LB will show growth as ampicillin will be present killing bacteria but no glowing because no arabinose will be present for glowing to be activated, the same result will be seen in the plate containing +pGLO, LB and ampicillin. The plate with –pGLO, LB and ampicillin will show no growth and no glowing as no arabinose is present for glowing to be activated
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.
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...
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.