+ Pglo And Pglo Lab Report

E. coli HB101 was transformed with pGLO plasmid then grown on media containing ampicillin and/or arabinose and on medium containing neither (Brown, 2011). This is done for selection of transformed cells since not all cells are expected to take up the plasmid (Brown, 2011). We also expect roughly the same CFU on any plate(s) receiving samples from the same microcentrifuge tube, since they are getting the exact same

First, 50 uL of bacterial cells were made competent by being treated with 590 uL of CaCl2 in three different test tubes before transformation of the plasmids took place. In order to enhance the uptake of the plasmid DNA the instructor added the CaCl2 solution to the bacterial cells. The tubes were placed into a cold ice bath for about ten minutes after the CaCl2 solution was added into the test tubes of E. Coli.

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

13. Use a sterile plastic loop to distribute the cell suspension evenly on the plate by “skating” the loop back and forth across the LB agar plate several times.

How does the addition of pGLO plasmid to a solution containing E. coli bacteria affect the growth and characteristics of the bacteria? Genetic transformation is the incorporation of foreign DNA into an organism to potentially change the organism’s trait. Plasmids are small circular DNA that replicate separately from the bacterial chromosome. In nature, these plasmids can be transferred between bacteria allowing for the sharing of beneficial genes. Due to this characteristic, plasmids allow for genetic manipulation and can be moved between bacteria easily. The pGLO plasmid utilized in this experiment encodes the gene for Green Fluorescent Protein (GFP), which under the right conditions can produce a glow. The gene regulation system present in the pGLO plasmid requires

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,

Froger, Alexandrine, and James E. Hall. "Transformation of Plasmid DNA into E. Coli Using the Heat Shock Method." Journal of Visualized Experiments ser. 2007.253 (6) Web. 22 Nov. 2016.

To start the experiment two small plastic tubes were labeled negative pGLO and positive pGLO as the first step in this experiment. A pipet was then used to move 250 microliters of calcium chloride into each tube and the tubes were iced. Bacteria was added using a new sterilized loop each time to the positive and negative pGLO tubes. The loop was twisted around in the tube to ensure that the bacteria was evenly mixed into the solutions and the tubes were iced once again. Another loop was dipped into the tube containing the plasmid and it was removed when there was a visible slight film of residue. The plasmid was dipped into the tube that was labeled positive for the pGLO gene. The two tubes labeled positive pGLO and negative pGLO were iced

As expected the higher growth was seen in the E.coli cells growing outside ampicillin environments, this is because they grow freely with only minor risk, such as contamination. Possible errors in the experiment can have included the difference in the amount of liquid broth added to the E.coli sample that contained no plasmid and that which contained a plasmid. The sample containing no plasmid received half the amount of liquid broth (150µl) as that of the other two samples (300µl). Liquid broth can have incremented the growth in the plates containing plasmid twice as much as on the rest of the plates. Another artifact which can have affected the results was the time each solution spent in both the ice and hot water bath. Uneven sharing may have also taken place within the E.coli used for each plasmid leading to different amounts of colonial growth in the different agar plates used. More accurate results would have been possible if there had been more variation in both E.coli and plasmid

Purpose: The purpose of this experiment is to teach the students step by step on how the genetic transformation process works using pGLO. Genetic transformation is when an individual cell’s genetic material is changed by exogenous DNA.

The objective of this experiment was to genetically transform E.coli cells to express ampicillin resistance and to produce the green fluorescent protein by using the pGLO plasmid. It was hypothesized that only the cells with pGLO DNA added to the solution would be able to survive in the same environment as the ampicillin, and that only the cells grown in the plate with arabinose would fluoresce bright green. The results supported the hypothesis, showing that the +pGLO LB/amp/ara plate was the only plate that had fluorescent cells and grew. Additionally, that +pGLO cells were the only ones that grew on an LB/amp plate.

Hagen M, Ashraf M, Kim I, Weintraub N, Tang Y (2018) Effective regeneration of dystrophic muscle using autologous iPSC-derived progenitors with CRISPR-Cas9 mediated precise correction. Medical Hypotheses 110:97-100

\textbf{A positive feedback mechanism to maintain the polarity patch on the plasma membrane.} \textbf{(a)} Cdc42 exists as GTP-Cdc42 (ON state) or GDP-Cdc42 (OFF state). The GTP-Cdc42 indirectly converts neighboring GDP-CDC42 into GTP-Cdc42, which sets off a positive feedback loop. GTP-Cdc42 exists predominantly in the plasma membrane and diffuses very slowly while GDP-Cdc42 diffuses fast in the cytosol. Since one of the components (the scaffold protein Bem1) that goes into causing the positive feedback gets depleted, the patch of GTP-Cdc42 stops growing. \textbf{(b) and (c)} Actin cables deliver patches to dilute the polarity patch to make it wander.}

Tissue regeneration through cell differentiation from one cell type to another is a phenomenon occurs in some species of fish and amphibians, however, mammals are incapable of reprogramming one cell lineage to achieve a similar result. Specifically, in human and mouse wound healing procedure normally generate scar with high collagen deposition, regenerated skin which lacks some features such as hair follicle and cutaneous fat. A recent study shows that a large skin wound in a mouse model is capable of regenerating hair follicles under the control of the Wnt and fibroblast growth factor (FGF) pathways. (1) In this article, Plikus et. al. discovered in the same mouse model; during wound healing process, cutaneous fat can be

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