Carson Adams 1-23-12 Mrs.

Miller AP Biology Lab 6 (12/14/12) Question- Will the introduction of gene plasmids that contain information for a certain anti-biotic resistance into a currently non-resistant bacteria culture result in resistance through the adoption of the plasmid? Hypothesis- The plasmid being introduced will be grafted into the DNA of the bacteria, resulting in the creation of the proteins necessary for resistance to a certain antibiotic. This will therefore genetically alter the susceptible bacteria to be resistant to the affects of the antibiotic. Objectives- The objective of this experiment is to evaluate the ability of bacteria to be genetically altered, as well as to understand the basic genetic concepts of DNA, alterations to it, and how they affect protein synthesis within a cell. Independent Variables-Plasmid introduction -Use of antibiotic -Time to grow Dependent variable-Bacterial growth Controls-Type of growth stimulant used -Type of antibiotic used -Type of bacteria used -Temperature Materials 2 2 2 1 2 4 1 1 mL 1 mL .02 mL 1 1 1 Luria agar plates Luria agar plates with ampicillin Microcentrifuge tubes Inoculating loop Bacti-spreaders Sterile graduated syringes Capillary micropipette with plunger Calcium chloride Luria Broth pUC8 solution E. coli plate Thermometer Water bath Procedure

-Refer to Wards 2002 Transformation of E. coli with pUC8 Lab Activity Student Study Guide for complete lab procedure Data -See attached page entitled Analysis for original data recordings and calculations Data Analysis In this experiment, growth was achieved to varying degrees on all four plates. Firstly, the luria agar + plate, the plate with transformed bacteria and no ampicillin, grew 55 colonies. Many of these colonies exhibited UV fluorescence, indicating that those colonies of bacteria grew from transformed bacteria, bacteria that had responded and adopted the plasmid for ampicillin resistance. On the luria agar plate, a lawn of bacteria (not able to distinguish individual colonies) was grown. They showed no signs of UV fluorescence. The difference in colony amount between the two luria agar plates with no growth inhibitors was likely due to the cell trauma incurred by the polar-bearing of the + bacteria in order to place them in competent state. This cold and hot flash likely resulted in the death of many cells, leaving fewer founders to begin colonies than there were in the - bacteria plates. Secondly, 35 colonies were observed on the luria agar with ampicillin + plate, whereas only 12 colonies had grown on the other plate with ampicillin present. The 12 colonies that grew, even in the presence of ampicillin, an anti-biotic, were either slightly naturally resistant, or were isolated from the affects of the drug by the death of other bacterial E. coli which had, to some degree, broken down ampicillin, allowing other bacteria in the center of clumps to grow. These colonies showed no UV fluorescence. Many of the 35 colonies that grew on the ampicillin, however, did. This indicates and explains why these colonies survived in the presence of the antibiotic. Not as many colonies grew on the ampicillin plates as they did on the regular agar plates, regardless of transformation though. This is likely because the transformation procedure isnt 100% efficient, leaving some bacteria dead or untransformed. These cells would therefore not grow, leaving fewer viable, resistant founders to create colonies. Conclusion The original hypothesis that transformation of the E.coli with pUC8 would result in ampicillin resistance proved to be true from the experiments carried out. This is implied by the fact that far fewer colonies of untransformed E. coli were grown on an ampicillin plate than transformed E. coli. This shows that transformation gave the E. coli a better chance of surviving in the presence of ampicillin than it would have, meaning it had some resistant qualities. From this experiment, we can also conclude that transformation is not complete or perfectly effective. Not all of the bacteria that had been exposed to pUC8 accepted and began coding for the new gene. If that had been the case, the number of colonies on the regular luria agar + would be equal to the ampicillin agar +. This is not so, because the ampicillin killed or stopped the growth of all the bacteria that had not been transformed, leaving only a fraction of the original bacteria, leaving only the ones that had been transformed, and therefore made resistant. As a side-note, it can also be observed that, to some degree, the process by which this experiment creates competent state, or the state allows bacteria to accept plasmids, might kill or disable a portion of the bacteria involved. This can be seen by the fact that while a lawn was grown on an inhibiting medium with no transformation, only 55 colonies were grown from the

group of bacteria that had been transformed by heat baths and flash coolings on the same substance. Questions -11 Questions (Refer to the Assesment page on the alternate side of the aforementioned data table page) Abstract This experiment demonstrated that the transformation of a cells DNA can occur by introducing a new plasmid into the cell, and that this plasmid will be treated as regular DNA and be coded for to create proteins that change the behaviors or traits of the cells. In this specific instance, pUC8, a plasmid which causes the acceptor to become resistant to the effects of ampicillin, was introduced to bacterial E. coli through rapid heating and cooling. This bacteria was then placed on both a regular luria agar plate and an ampicillin agar plate in order to see if transformation would allow the bacteria to grow on the anti-biotic. Then, two untransformed samples of E. coli were placed on similar plates in order to prove that without the plasmid, that E. coli could not survive on the ampicillin. This was also done to examine the effects of transformation or the method of polar-bearing (heat/cold flashing) on bacteria. These were then left to incubate and results were recorded. Minimal growth was seen by the non-transformed bacteria on the ampicillin plate, showing that E. coli cannot grow on ampicillin. However, reasonable growth was observed on the ampicillin plate that housed the transformed bacteria. This showed that many of the bacteria that had been exposed to pUC8 had received the plasmid and had been transformed to be resistant to the effects of the anti-biotic ampicillin. It was also observed that growth on the normal plates differed despite no difference in environment. Less growth occurred on the regular agar plate that housed the transformed bacteria. This is likely due to some cell death as a result of polar-bearing. Basically, it was observed that transformation can occur and can change the way in which cells act or produce proteins.