E.coli Lab Report PDF

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Erika Martinez Bio 223 Lab Report: The growth and transformation of E.coli Introduction: Bacteria are classified as prokaryotes. Prokaryotes are single-celled organisms such as bacteria that have no distinct nucleus. (Introduction to Bacteria) A typical prokaryote is bound by a plasma membrane and a cell wall. (Prokaryotes) The fact that the genetic material of bacteria is contained in a single, circular chain of Deoxyribonucleic acid that is not enclosed with a nuclear membrane. Deoxyribonucleic acid (DNA) is a natural polymer which encodes the genetic information required for the growth, development, and reproduction of an organism. Found in all cells, it consists of chains of units called nucleotides. E. Coli is bacteria. (Deoxyribonucleic Acid) Eschenchia coil is the full genus and species name. Competent E. Coli cells are cells that are able to pick up the plasma. We use competent E. Coli cells in our experiment because we want to the E. coli to pick up extracellular DNA. Ampicillin is penicillin antibiotic to treat bacterial infections. The reason why we use ampicillin is to select the bacteria that has the plasmid which has the antibiotic resistance gene. A plasmid is a small DNA molecule within a cell that is physically separated from a chrosome's DNA and can replicate independently. Transformation involves the uptake of free or naked DNA released by donor by a recipient. (Introduction to Bacteria) The purpose of this experiment was to observe the transformation and growth of E. Coli cells on Luria broth and Luria broth with ampicillin. Materials and Methods:

The materials needed in this experiment were an alcohol, starter plate of E. coli, 2 x 1.5 mL tube of the same color, ice bucket with glass vial of calcium chloride, 100 µL pipette and tips (yellow) 1 mL pipette and tips (blue), 1 yellow plastic inoculation loop, room temperature Luria Broth (glass vial), 2 LB media plate, 2 LB + amp plates, sharpie, and spreader. Mark one of the 1.5 mL tubes as “+” and the second as “-“. These will indicate which of your E. coli has the plasmid (+) and which does not (-). These tubes should be kept on ice. Add 250 µL of cold Calcium Chloride (CaCl2) to each tube. Using a yellow plastic inoculating loop, transfer some of the E. Coli colonies from the starter plate into the 1.5 mL tube labeled (+). Be careful not to scrape any agar off the plate, you just want the E.coli. Dip the inoculating loop into the Calcium Chloride in the 1.5 mL tube and stir vigorously from side to side but be careful not to splash any of the liquid out. Before removing the inoculating loop, check to make sure the mass of E. coli cells has detached and is. Mix the E. coli cells into the Calcium Chloride by pipetting up and down. There should be no visible clumps of cells in the tube when you are finished. Place the tube back on ice. Repeat this same process with the (-) tube. Both tubes should be on ice when you’ve completed this step. Add 10 uL of plasmid DNA to the (+) tube. The plasmid DNA is on ice. Gently turn the tube upside down twice to mix and tape the tube so that all the liquid is in the bottom. Return tube to ice and incubate for 15 minutes. While you wait, label one LB + amp plate “+” and the second “-“. When 15 minutes has passed, it is time to heat shock the E. coli in the 1.5 mL tubes. Carry your group’s ice bucket over to the heat block and place your tubes directly from the ice into a spot on the heat block. Incubate for 90 seconds at 42oC. When the 90 seconds is up, immediately remove the E. coli from the heat block and place back on ice for at least 1 minute.

After at least 1 minute has passed, add 250 uL Luria Broth to each tube. Gently turn the tube upside down several times to mix. Incubate both tubes at room temperature for 15 minutes. Now it is time to transfer the E. coli to the correct plates. Gently pipette 100 µL of the correct E. coli to each plate. Keep the E. coli in one spot, do not let is splash around the plate .E. coli labeled “+” go on the plates labeled “+” LB amp plate . E. coli labeled “-” go on the plates labeled “-” LB amp plate. E. coli labeled “-” Also goes on the (+) LB plates and LB media goes on the (– )LB plate. The E. coli must now be spread on each plate. The metal spreader must be sterilized between plates. All of this must be done near the flame to keep the environment sterilize. Dip in alcohol and let excess liquid drip off before holding spreader over the flame. Allow alcohol to burn off and keep the spreader in the flame for at least 10 seconds. Move the spreader to just outside the flame and fan to cool. Gently touch the spreader to part of the plate where there is no E. coli. Move spreader around plate to cool, before beginning to spread the E. coli. Once cool, move the spreader over the E. coli and move around the entire plate. Avoid the very edge of the plate and make sure liquid is well spread so that it won’t drip when we turn the plates upside down in the incubator. Once E. coli is spread on all the plates, make sure to label with the initials of all group members. The plates are now ready to go into the incubator. Results: The LB plate negative control had no growth on it. The LB plate positive control had growth on it. The growth on the LB plate positive control was high. The LB with amp plate negative control had no growth on it. The LB with amp plate positive control had growth on it. The growth on LB and amp positive control was medium.

Table 1: LB Plates Negative control No growth LB stands for Luria broth.

Positive control High growth

Table 2: LB with amp Plates Negative control Positive Control No growth Medium growth LB stands for Luria broth and amp stands for ampillcin. Discussion: There was no growth on the LB plate negative control because there were no E. Coli cells. The LB plate positive control had growth on it because it was full of nutrients. The growth on the LB plate positive control had a high growth because it contained no ampicillin so that ampicillin didn't select the transformed bacteria. The LB plate with amp plate negative control had no growth on it because the ampicillin killed all the E. coli cells. The E. coli cells were killed by the ampicillin because it wasn't resistance. The LB with amp plate positive control had medium growth on it because the ampicillin selected the transformed bacteria. The error made in this experiment was the negative controls were both contaminated because the experiment wasn't close enough to the flame. In the future, to prevent this error from occurring happening the flame would be closer to the experiment to keep the environment stable.

Works Cited Page "Deoxyribonucleic Acid (DNA) - History, Structure, Function, Replication Of Dna, The Genetic Code, Expression Of Genetic Information." Cells, Genes, Cell, and Proteins - JRank Articles. N.p., n.d. Web. 09 May 2017. . "Introduction to Bacteria" Carolina biological supply company. Web. 2012.

"Prokaryote." Prokaryotes, Bacteria, Cells, and Organisms - JRank Articles. N.p., n.d. Web. 09 May 2017.....