What Is A Transposon?

Abstract In this lab of transforming bacteria that was experiment today, I will be identifying the process of bacterial genetic transformation and how to calculate transformation efficiency. The samples that will be used in today’s bacteria will contain samples of E.coli sand inserted DNA plasmid into their genetic sequence. If done correctly the results will show a successful genotypic and phenotypic mutations, which will display fluorescent under ultra-violent lights or show signs to being resistant to ampicillin. This experiment was primarily for the purpose of growing E. Coli bacteria.

70µL of competent E.coli are added to both test tubes; pUC18 and Lux (Alberte et al., 2012). Both test tubes are then tapped and placed back into the ice bath for 15 minutes. While waiting, another test tube is obtained, filled with 35µL of competent cells and labeled NP for no plasmid. A water bath is preheated to 37 degrees Celsius and all three labeled test tubes are inserted into the bath for five minutes (Alberte et al., 2012). Using a sterile pipet 300µL of nutrient broth are inserted into both the control and Lux test tubes and 150µL are inserted to the no plasmid test tube to increase bacterial growth. All three test tubes are then incubated at 37 degrees for 45 minutes. Six agar plates are obtained and labeled to correspond each test tube, three of the plates contain ampicillin. A pipet is used to remove 130µl from each test tube containing a plasmid and insert it into the corresponding agar plate. For this, a cell spreader is first

The purpose of this experiment is to make E.Coli competent so that it can be transformed in order to become immune to ampicillin, then we would be able to determine the transformation efficiency of the culture. We determine this by preparing 4 plates of E.coli, each labeled “LB-plasmid”, “LB+plasmid”, “LB?Amp-plasmid”, and “LB/Amp+plasmid”. This meant that either should have lacked plasmid and Ampicillin, with plasmid but lacked Ampicillin, without plasmid but with Ampicillin, or were with Ampicillin and plasmid, respectively. Then we made the bacterial cells competent by adding CaCl2 to 2 vials of the colony (one with plasmids), and incubating on ice, then heat shocking, and returning to ice. Luria Broth is then added and left to sit for 5-15

This pBlu lab had for purpose to present the changes of the strain of E. coli bacteria due to new genetic information being introduced into the cell. In this experiment we are freezing and heat shocking the E. Coli bacteria that is then forced to take the plasmid DNA. The E. coli then transforms the pBLu plasmid, which carries the genes coding for two identifiable phenotypes. After following the Carolina Biological steps our lab worked well and we able to see some colonies of bacteria on the plates. The x-gal plate showed a significant amount of bacteria to confirm that the pBlu plasmid took over the E. coli strain.

The LB Plate acts as a control for the LB/AMP because it shows that the bacteria without the plasmid, but still went through the process, survived. The second plate, which contained LB/AMP, left no trace of bacteria behind. This is because the AMP is an antibiotic, which kills off the existing bacteria (LB). This plate also acts a control because without the plasmid, the bacteria can’t grow in the presents of Antibiotics.

There of the dishes turned out as expected in this experiment. Our group expected there to be growth in the LB -pGLO dish as the bacteria were not exposed to the antibiotic ampicillin. Furthermore, our group also expected to see inhibited bacterial growth in the LB/amp +pGLO dish as there was ampicillin in the dish, but some of the bacteria were immune as they possessed immunity to the ampicillin. Moreover, our group expected that there would be no bacterial growth in the LB/amp -pGLO dish, as the bacteria were exposed to ampicillin and were not immune. However, the final dish, LB/amp/arbo +pGLO, did not turn out as expected. While it was expected to allow for inhibited bacterial growth and the bacteria to become florescent,

While the plate with -pGLO on the LB/AMP agar plate would not have any colonies, and the –pGLO on the LB agar plate would have many colonies of E. coli. The +pGLO on the LB/AMP plate did have two colonies but the only colonies produced on the LB/AMP/ARA plate had a colony not growing on the actual agar plate. The –pGLO on LB/AMP did not have colonies of E. coli growing on it. But the –pGLO on the LB agar plate had many colonies growing on the plate. All of the colonies were clear in color even under the ultraviolet light. Therefore the plate that did not support my hypothesis was the plate with the arabinose sugar, since that was the one thought to grow E. coli do to the ampicillin resistant DNA that was in theory picked up by the E. coli. This plate also should have glowed green under the UV light but instead the colonies remained clear. I hypothesize that E. coli cells recognized the foreign DNA and then destroyed it believing the new DNA would be harmful to the cell. This tendency of bacteria cells to kill foreign DNA could answer be a problem that would explain why we had few colonies that survived in the presence of ampicillin and why none of the colonies in the arabinose glowed green. Also the time the E. coli

The experiment was a success and the four E Coli plates brought forth the expected results. The two control plates were -pGLo LB and -pGLo LB/amp. On the -pGLo LB plate, there was no ampicillin to prevent the growth of E Coli so the bacteria thrived and grew massively across the plate. There were no specific colonies, rather there was E Coli spread out across the entirety of the gel. This serves as a control because it proves that E Coli was able to grow in the environment provided for it.

In this experiment the objective was to transform E. coli with the pGLO plasmid and calculate the transformation efficiency. The hypotheses were that the plate with only LB agar and untransformed E. coli would grow a lawn; the control plate of untransformed bacteria with LB and ampicillin would experience no growth; the transformed plate with just LB and ampicillin would grow colonies of bacteria but it would not glow green under UV light; and the transformed plate with LB, ampicillin and arabinose would grow colonies that would glow green under UV light. The results found supported each of these hypotheses as the bacteria grew as predicted. The

At the same time, bacterial colonies are needed to be placed into PCR, and use photograph of gel to determine the size of tetracycline resistance genes to distinguish the bacteria. It is relevant to research 1 because serial dilution is the first step to get colonies' samples that is further being used to distinguish plasmids. It is also relevant to research 2 because serial dilution is also the first step to count the frequency of tetracycline resistant. Week 2: Experiment Part B By placing the bacteria into the PCR machine, much more copies of genes would be made to make them visible under electrophoresis, which can then be compared with control ladder to distinguish the kind of bacteria.

The unknown project was an experiment where the student was given a petri dish of unknown bacteria. Tests were performed on it to identify the specific species. The purpose of the experiment was to learn about the identifying tests, and procedures in the identification of specific microbes. The reason the master plate was used to create a working plate is so that if the working plate becomes contaminated, one can resort back to the master plate for the pure strain of the bacteria and create a new working plate. The purpose of the first procedure, the gram stain, was to be able to dye and then distinguish gram negative and gram-positive cells on a smear. The second procedure, the citrate test is used to see if the bacteria can use citrate as

a. LB+plasmid and LB-plasmid: Both of these plates had a lawn of bacteria. This proves

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.

Both the LB/amp plates (LB/amp –pGLO and LB/amp +pGLO) should be compared. Comparing the results on each plate would show whether or not the bacteria was transformed. Bacteria with pGLO added (LB/amp +pGLO) should be able to grow in the presence of ampicillin because

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].