Project Information

Grade Category: 10-12
Team Size: 3 to 6
Subject Area: Biology
Project Type: Experimental
Language: English
Software Tools used: EditPad Lite and Photoshop CS2
Hardware Tools used: Digital Camera
Source of the Idea for the project: We originally wanted to investigate the danger of transformation in soil from GM plants to bacteria. However, due to insufficient lab equipment, and pathogenic bacteria present in the soil, we changed our focus to this topic, which can still be related to the transformation in soil. As already stated, if we can find an optimal pH for bacteria to transfer DNA, it is possible to alter this pH in soil in order to limit the frequency of transformation, hence reducing the risk of transformation DNA from GM plants to bacteria in soil
Special Skills used to build the project: HTML, XHTML and CSS
Awards won for this project:

Problem

How does extracellular pH affect the genetic transformation frequency of calcium treated Escherichia coli?

Abstract

The effect of extracellular pH on the frequency of genetic transformation in calcium-treated Escherichia coli was investigated, focusing on whether pH affects transformation frequency through altering components of the proton motive force, membrane potential and pH gradient. Optimization of transformation frequency will allow laboratories to more efficiently replicate plasmid DNA. The transformation frequency of soil bacteria with genetically modified plant transgenes, and of bacteria in clinical settings, may be reduced by the avoidance of optimal pH conditions.

E. coli were made competent and transformed with an ampicillin resistance carrying plasmid, in solutions of different pH ranges. Transformants were selected for ampicillin resistance. In the first and second experiments, transformation frequency peaked at pH 6.88-7.14, while in the third experiment transformation frequency peaked at pH 7.85-8.18. The optimal pH for E. coli transformation is concluded to be about pH 7.4, supporting Norgard et alís optimal pH range of 7.25-7.75. E. coli should continue to be transformed within this pH range until experiments testing smaller pH ranges can suggest a more specific optimal pH. The optimal pH range is very similar to the intracellular pH of 7.5. This similarity suggests that the membrane proteins involved in transformation and exposed to both intracellular and extracellular pH levels function most efficiently at the intracellular pH. Thus, these proteins function best when extracellular pH matches intracellular pH.

A comparison of the experimental transformation frequencies to literature values of the magnitude of components of the proton motive force at the same extracellular pH levels indicates that no component of the proton motive force is involved in E. coli transformation. This does not support the existence of an electrogenic calcium-DNA symport mechanism in E. coli Other possible mechanisms and driving forces of DNA translocation into E. coli should be investigated further. The impact on transformation frequency of very acidic extracellular pH levels, and of pH variation at specific stages of competence development, continue to be investigated.