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Bacterial Transformation Lab

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Lindsay Young

on 9 December 2013

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Transcript of Bacterial Transformation Lab

Biotechnology Bacterial Transformation Lab: The effects of pGLO DNA on E. coli
Method
Introduction
Bacteria transformation is the process of a bacterium absorbing and integrating naked DNA located on the surface of their membrane. This usually occurs with plasmids, small circular molecules of DNA. The theory we have learned in class is that the naked DNA is then integrated into the bacterium's DNA, causing the expression of new traits. The bacteria we will be using in this lab is called "pGLO", which is a bacteria with a gene for resistance to ampicillin (an antibiotic that eliminates bacterial growth by destroying cell walls), an arabanose sugar and Glowing Fluorescent Proteins (GFP's). The arabanose sugar located in the bacteria allows for the production of GFP's (glowing florescent proteins) through the "operon" system. This occurs because in order for RNA polymerase to synthezise the regulatory genes (in this case, the GFP's), the "repressor" must not be binding to the operator. The presence of the arabanose sugar negates the repressor by binding to it and causing a conformational change - now, the bacteria can produce the GFP's. Because of this, only plates cultured with the arabanose sugar ("ara") will have glowing cultures.
Results
Discussion
In this experiment, we discovered quite a few of our bacteria plates did not produce the expected results. A pattern we observed was that the plates that were supposed to have pGLO incorporated into their e. coli's DNA apparently did not - we can see this because in the LB/amp (+) and the LB/amp/ara (+) plate, there was no growth at all, when in theory the ampicillin resistant gene found in the pGLO should've resulted in bacteria growth.
Abstract
Bacteria are microscopic, single-celled organisms. In this experiment, we will demonstrate and explore how to transform bacteria with new DNA so that it expresses different genetic information. The purpose of this lab is to explore the effets of bacterial transformation on e. coli bacteriums. We will be inserting pGLO DNA into the genome of the E.coli bacteria through the use of innoculation loops, Laurel Broth, "Transformation Solution" and procedures such as heat shock and incubation. Our belief is that the bacterium with pGLO and arabanose sugars injected into it will produce glowing bacteria colonies. We observed that only the LB (pGLO -) plate had growth on it. Because of this, we concluded that the pGLO DNA did not incorporate into the e. coli DNA, because if it had, there would have been traits expressed - resistance to ampicillin, growth of glowing colonies - on the other slides.
Our alternate hypothesis for this experiment will be that the pGLO DNA will incorporate in the e. coli DNA and produce new traits. THe Lb/amp (pGLO -) plate will have no growth, the LB (pGLO -) plate will have a "lawn" of growth (meaning colonies covering the entire surface), the LB/amp (pGLO +) plate will have a number of colonies acoross the surface, and the LB/amp/ara (pGLO +)plate will have a number of glowing colonies.

Our null hypothesis for this experiment will be that the pGLO DNA will not incorporate into the e. coli DNA. Because there will be no change in the DNA, the bacteria will still be killed by the ampicillin, so therefore the LB/amp (pGLO -), LB/amp (pGLO+) and the LB/amp/ara (pGLO +) will have no growth in them. The LB (pGLO -)plate will have a "lawn" of colonies growing across the surface, due to the Laurel Broth.

Our prediction is the alternate hypothesis will be correct, and that the pGLO DNA will incorporate into the e.coli and produce new traits.
The purpose of this lab is to understand how transformation occurs, as well as the biological results and consequences that come from transformation; and to understand the importance of transformation in prokaryotic (and eukaryotic) cells.
In the LB/amp (pGLO positive), we expected to see colonies growing, because the bascteria absorbed the plasmid that was resistant to ampicillin - instead, there was no growth. In the LB/amp/ara (pGLO positive), we expected to see glowing colonies, because this plate had the arabose sugar in it to activiate the operon - instead, there was no growth of any kind. Both of the two pGLO positive slides were effected because we did not properly "heat shock" the cells, resulting in ineffective growth.
Conclusion
The purpose of this lab is to understand how transformation occurs, as well as the biological results and consequences that come from transformation. We discovered that there were no growth on our LB/amp (pGLO -), LB/amp (pGLO +) and LB/amp/ara (pGLO +) plates, and about 320 white, circular colonies growing on the LB (pGLO - ) plate. Our results did not support the theoretical science we learned in class, or other experiments done in this field of study. Our results do relate to the field of study in the way that if bacteria is not properly introduced to new plasmids, it will be unable to incorporate the plasmids DNA into their DNA.
Discussion
LB/amp
Expected Results:
no growth
LB (pGLO negative)
Expected results:
"lawn" of bacteria growth
LB/amp (pGLO +)
In the LB/amp plate, there was no growth of bacteria because of the presence of the ampicillin. However, this is not supported by scientific theory, because the ampicillin resistant gene in the pGLO should have been able to couteract the ampicillin, for the most part. Because of this, we can conclude that the pGLO genetic information was not properly transferred to the genetic makeup of e. coli
LB/amp/ara (pGLO +)
Expected results:
glowing circular cultures
Observed Results:
no growth
In the LB/amp (pGLO negative) plate, there was no growth observed because of the presence of ampicilin, which is an antibiotic that kills the growth of bacteria. This is supported by scientific theory and previous experiments, because amplicllin destroys the membranes of cells, making it impossible for them to survive and reproduce.
Observed results:
about 320 small, white circular colonies that grow predominantly towards one side of the plate
In the LB (pGLO negative) plate, there was substantial bacterial growth because of the Laurel Broth, which acts as a food for bacteria. In theory, the bacteria should have covered the entire surface of the agar because of the presence of food for it, and also because of the heat incubation. However, the bacteria did not cover the entire surface of the agar, most likely due to the improper spreading of the culture across the gel prior to incubation (using the innoculation use)
Expected results:
growth of colonies on surface
Observed results:
no growth
Observed results:
no growth
In the LB/amp/ara plate, there was no growth of bacteria because of the presence of ampicillin. However, this is not supported by scientific theory, because the ampicillin resistant gene in the pGLO should have been able to couteract the ampicillin, for the most part. Because of this, we can conclude that the pGLO genetic information was not properly transferred to the genetic makeup of e. coli. Because of this, we cannot tell whether the bacteria wouldve been "glowing" as a result of the arabanose sugar or not.
Conclusion
Several changes couldve been made to this lab so that the results were more effective. We should have placed the microtublues in a foam "floater" even when they were in the ice, as so to avoid water/ice getting into the samples and contaminating them. We should have made sure to collect enough/any of the bacteria to put into the solution. We should have made sure that the microtubules stayed completely immersed in ice in the second ice bath, as so to ensure proper "heat shock". We also should have made sure to spread the bacteria thouroughly around the surface of the bacteria using the innoculating loop.
To start this lab, you must first label your micro tubes accordingly - one with - plasmid, one with +plasmid. You must then pipette 250 uL of the transformation solution (CaCl2) into each. Using a sterile innoculation loop, scoop up a single colony of e. coli bacteria from the starter plate. Immerse the loop in the CaCl2 liquid in the micro tube marke + plasmid, and spin the loop until all the bacteria is incorporated into the solution. Repeat this step with the - plasmid micro tube. Using a pipette, transfer 10 uL of the plasmid solution directly into the + plasmid tube. Tap the tube lightly with a finger to mix. Place both tubes onto ice, and incubate for 10 minutes. While waiting, label the agar plate lids with LB/amp (-pGLO), LB, LB/amp (+pGLO) and LB/amp/ara. After 10 minutes, place the tubes into a test tube float and put tubes into a water bath (at 42 degrees celcius) for 50 seconds. After 50 seconds, immediately put the tubes back on ice for two minutes. Transfer 250 uL of LB nutrient broth to both tubes, making usre to use a new sterile pipette tip each time. Use a pipette with a new sterile tip to transfer 100 uL of each onto the according plates. Using a new sterile inoculation loop, methodically and thouroughly spread the solutions across teh agar. Stack the plates and tape them together. Place the stack upside down in a 37 degree culcius incubator for 24 hours.
LB/amp (pGLO negative)
No growth was observed
LB (pGLO negative)
a large number of white, circular colonies were found across the surface of the agar
LB/amp (pGLO positive)
no growth observed
LB/amp/ara (pGLO positive)
no growth observed
Results
Conclusion
Because of our results, we must reject our alternate hypothesis, and accept the null hypothesis. The e. coli bacterium exposed to the pGLO appeared to not incorporate it into its DNA, as did not express the expected results and traits.
Refrences
Reece, Jane B., and Neil A. Campbell. "Genetics - Biotechnology". Campbell Biology/Jane B. Reece ... [et al]. San Fransisco, CA: Benjamin Cummings, 2011. 398. Print.
Anderson, Mr. "AP Biology Labs - Part 2"
YouTube
. YouTube, 10 May 2013. Web.
AP Biology Investigative Labs Manual (College Board, 2012), Investigation 8 - Biotechnology: Bacterial Transformation
"Bacterial Transformation."
YouTube
. YouTube, 15 Oct. 2012. Web. 08 Dec. 2013.
Student Manual: pGLO Transformation
purpose statement:
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