How Escherichia Coli To Form A Recombinant Plasmid

Plasmid map of pRSETB. Primers were designed to amplify via PCR to cDNA. The PCR product was digested with XhoI and EcoRI enzymes and ligated into the pRSETB plasmid. The pRSETB plasmid contains a T7 promoter region, is ampicillin resistant, inducible with Isopropyl β-D-1-thiogalactopyranoside IPTG, a molecular mimic of a lactose metabolite that triggers transcription of the lac operon, and has XhoI and EcoRI cut sites. The pRSETB plasmid is transformed into dH5α E. coli and plated on carbenicillin plates. Colonies are selected and grown on a carbenicillin plate while PCR is used to check that the plasmid that was up-taken was not

After analyzing the data recorded for both the agar plates containing ampicillin and those that did not, it can be concluded that the data provides enough evidence to reject the null hypothesis. There is enough evidence to support the alternative hypothesis stating that there is a correlation between plasmids coding for an antibiotic resistant gene and bacterial growth in ampicillin. When a bacterial solution containing either pUC18 or the lux plasmid is transformed in an agar plate containing ampicillin, only those cells which took in the plasmid are able to survive and replicate, forming individual colonies. Not all cells are transformed though, the chances of a successful transformation were extremely low. In order to see which cell transformed the cells were tagged according to their plasmid, in the plates containing pUC18 the only

This experiment was completed to conduct bacterial transformation and introduce ampicillin resistant DNA plasmid to E. coli bacterial cells. Observations are made of the difference in cell growth due to various environments provided within agar

The objective of this experiment was to observe the transformation occurrence with E.coli and the ampicillin resistance gene.When plasmids like lux or pUC18 are added in E.coli, they are more likely to survive in certain environments that contain antibiotics. Plasmid can carry genes enabling bacteria like E.coli to survive in harsh conditions. This experiment displayed how plasmid work when inserted in E.coli with and without ampicillin. In certain agar plates, ampicillin was added with either the lux plasmid or the pUC18 plasmid resulting in colony growth. In other agar plates, no ampicillin was added when either the lux plasmid or the pUC18 plasmid was added resulting in colony growth. If growth occurred from the bacteria when it was added in the Ampicillin agar plates, then it was determined that the E.coli was transformed successfully and is expressing the Ampicillin resistant genes.

There are five stages to this experiment: pre-incubation, incubation, heat shock, recovery and growth, and selection. First, two micro test tubes were obtained and labeled “+” and “-”. These labels were representative of transformed (+) and untransformed (-) Escherichia coli (E. coli). 250 microliters of transformation solution (calcium chloride) was added to the test tubes and placed on ice. During the pre-incubation period a single colony was placed into the test tube labeled “+” and was completely dispersed in the solution. This was repeated for the tube labeled “-“ as well. Then the solution was returned to the ice. In the incubation period, the pGLO DNA solution was added to the tube labeled “+” but was not added to the tube labeled “-“. The tube was then returned to the ice and incubated for 10 minutes. While this was undergoing the process, four agar plated were labeled according to what they had been treated with. The plates were treated accordingly: plate 1 contained lysogenic broth (LB)/ ampicillin (amp)/ plasmids with a green fluorescent protein (pGLO); plate 2 contained LB/amp/arabinose sugar with pGLO; plate 3 contained LB/amp without pGLO; and plate 4 contained LB without pGLO. The next step was heat chock. Both test tubes were placed in a water bath that was set at 42°C for 50 seconds. After 50 seconds the test tubes were rapidly transferred back to the ice were they

Before plating the strains on agar plates, dilutions of the three strains of cells were prepared with LB broth.

The lids were then placed on the tubes, and both tubes were placed in ice. E. coli bacteria cells that had been grown on a media plate overnight, were then added to each tube and completely circulated throughout the solution using a sterile loop. Both tubes were then returned to the ice. The pGLO plasmid was then added to one tube and the other acted as the control, and had no pGLO plasmid added to the solution. Both tubes were then placed on ice for ten minutes, then both tubes were submerged in 42°C water for 50 seconds in order to shock the bacteria and allow the membrane to become more permeable. The tubes were then immediately removed from the bath and replaced in ice for another two minutes. Luria Broth (LB) was then added to both tubes and left at room temperature for ten minutes. The pGLO- solution was added to a LB nutrient agar plate and to a LB nutrient agar plate that had ampicillin present in it. The pGLO+ solution was added to a LB nutrient agar plate that had ampicillin added to it and to a LB nutrient agar plate that had ampicillin, as well as arabinose. All four petri dishes were stored inverted at 37°C for 24 hours. (Weedman

Bacterial transformation is the result that occurs when exogenous genetic material is inserted into a prokaryotic cell. The new genotypic makeup translates into a new phenotypic expression. In the case of the lab, the plasmid, pGreen was introduced to the experimental group. In both the control and the experimental group, the existing bacteria was E. Coli MM294. Plasmids are parts of DNA that carry genes. The particular plasmid that was used in this lab was pGreen, which carries the genes GFP and ampR. GFP codes for a protein that makes the bacteria a fluorescent green color and ampR codes for a protein that makes the bacteria resistant to ampicillin, an antibiotic. The fluorescent green color is a trait needed for identification while the ampicillin resistance is crucial for the survival for the bacterial colonies.

The results obtained in the bacterial culture plates were the following: In Plate # I, the bacteria grew as a lawn of bacteria, making it uncountable. In plate # II without pGLO plasmid, there were no bacteria, all of them died in the presence of ampicillin. In plate # II with pGLO plasmid, the bacterial culture lived and grew. In plate # III with pGLO plasmid and araC protein, the bacterial culture grew and glowed. The transformation efficiency is the number of colonies on LB/amp/ara plate divided by the amount of DNA on the plate. Figure 1 shows the culture plates under visible light, figure 2 shows the culture plates under ultraviolet light, table 1 shows the characteristics of the culture plates obtained after the experiment, and Table 2 shows the calculations done for finding the transformation

Today, we are using Biotechnology to by-pass nature 's random fashion of generating variation. We will be genetically engineering a new organism through a process known as transformation (transfer of genetic information between organism’s takes places via the movement of an extracellular piece of DNA). Bacteria are great for experiments for genetic manipulation simply because of how long they take to produce, their simplicity, and how easily recognizable they are. The bacteria that we will be using for this experiment is Escherichia Coli, also known as E. coli. Most of this bacteria genetic information is carried on small pieces of extrachromosomal DNA. The small DNA molecules are known as plasmids. Plasmids carry genes that allow bacteria to neutralize antibiotics that would typically kill them only one molecule in 10,000 can successfully transform a bacterium in the most favorable conditions in the laboratory, so for it happen in nature by itself is incredible. It is a true test of survival of

Bacteria have evolved so that they can transfer different strains of resistance to their own species and different species (Khachatourians, 1998). The organisms have genetic traits in the chromosomes and extrachromosomal elements for resistance to occur (Khachatourians, 1998). Resistance can occur from a mutation on the chromosomal genes, leading to antibiotic sensitivity. Khachatourians states that, “such mutations occur at a rate of one per million to one per billion cells”. He also explains that, “the extrachromosomal elements (plasmids and transposons) are smaller pieces of circular DNA, each equivalent in size to about 1% of a chromosome” (Khachatourians, 1998). When the bacterial organism acquires the resistance against the antibiotic administered and send it to other species of bacteria this is known as genetic exchange (Khachatourians, 1998).

Transformation of the pAMP to the E. coli showed growth in all plates that did not contain ampicillin, which was to be expected as E. coli with or without the ampicillin resistant DNA would be expected to survive in an environment that did not contain the ampicillin. Additionally, E. coli that was not transformed did not survive in plates containing ampicillin; a fact that we had already predicted as the bacteria has no genes protecting it from the deadly antibiotic. However, we found slightly different results when the bacterium that was transformed with the pAMP was cultured in plates (labeled A and B) containing ampicillin. While we did see growth on the B plate, we did not see any growth on plate A. To explain this, we also must take into account the fact that different concentrations of bacteria were used to culture each plate. Plate B was cultured with 100 µL of bacteria while plate A only contained 10 µL of bacteria and 90 additional µL of luria broth. The bacteria without the pAMP transformation acted as a control when we tested the

Bacteria are haploid organisms that lack the true nuclei of eukaryotic organisms. They contain one copy of each gene thus the genotype of bacteria can be deduced from phenotypes. Bacteria can reproduce asexually by means of binary fission and have the ability to transfer genes by several mechanisms: conjugation, transformation and transduction. Bacterial conjugation is a process that involves the transfer of genetic material between bacteria through a contact dependent process. The gene transfer is carried out from a donor cell that contains a conjugative plasmid to a recipient cell with no plasmid present. Plasmids are circular, double-stranded DNA molecules that can replicate independently of the host cell genome or can be integrated into the genome and replicate with it during cell division. Conjugative plasmids are present in both Gram-positive and Gram negative bacteria. The conjugative transfer of this bacterial plasmid encodes resistance of bacteria to antibiotics. Subsequently, conjugation and spread of antibiotic resistance represents a severe problem in anti-microbial treatment, particularly of immunosuppressed patients and in hospitals.

To control a spread of disease, antibiotic synthesized from a living organism is chosen as a solution because of capability in preventing infection (Edward Boden and Anthon Andrews, 2015). On the other hand, there are diverse bacterial strains that have an antibiotic resistance system. Using antibiotic may help bacteria to develop this ability by selecting unwanted survivor by accident which cause bacteria become more dangerous. In living animals, varied types of bacteria with resistant property live to gather and some can mate across their strain and transfer antimicrobial resistance plasmid gene through conjugation by using specific bridge called pillars (CAMMACK, Richard et al., 2006). The new strain will be occurring, and it might tolerance more than one antibiotic.

9. After the incubation, heat shock the cells. Do this by removing the tubes directly from the ice into 42degree Celsius water bath for 90 seconds.