Calculating Transformation Efficiency With E. Coli Bacteria and pVIB Plasmid

Abstract: Over the course of a few days the process of transforming E. Coli was tested using agar, luria broth, and ampicillin petri dishes. With a focus on aseptic techniques, the initial E. Coli colony was incubated without the presence of ampicillin. After sufficient growth, the E. Coli was transferred to test tubes and then manipulated using pVIB plasmid. Three new dishes, two with ampicillin and one without, were covered with the manipulated E. Coli and then incubated. The experiment resulted in a glowing colony on the LB Ampicillin + dish.

Introduction: In 1928, an experiment by Frederick Griffith suggested that bacteria are capable of transferring genetic information through a process known as transformation. Transformation is the genetic alteration of a cell resulting from the cell taking up exogenous genetic material from its surroundings through the cell membrane. Conjugation is the transfer of DNA from one bacterial cell to another bacterial cell. A plasmid is a circle of that transferred DNA that is distinct from the chromosome. Transduction is a process through which genetic material from a virus is inserted into a cell. Transforming E. Coli DNA with pVIB plasmid, when done correctly, results in a faint, glowing green colony. Calculated transformation is vital for repeated success in DNA transformation. Producing vaccines is becoming more important as the world grows in population, but more important population density. Bacteria, viruses, and parasites are adapting to scientists efforts to eliminate them, making it even more important that young scientists pursue microbiology.

Data:

Discussion:

What are you selecting for in this experiment?

We selected for the traits of luminescence and ampicillin-resistance. If bacteria glows, then it has taken DNA from the plasmid on its plate. If bacteria is placed on the same plate as ampicillin and survives, then we know that it also has taken up the plasmid.

What does the phenotype of the transformed colonies tell you?

The phenotype of the transformed colonies tells us that the plasmid DNA has been taken up by the bacteria and is being expressed by the transformed cells.

What one plate would you first inspect to conclude that the transformation occurred successfully? Why?

I would inspect the LB/AMP+Plasmid plate, because for the bacteria to transform and be ampicillin-resistant, it would have to be on the same plate as the plasmid. To show that it has actually transformed, it must be on the same plate as ampicillin, because if it grows on the plate with ampicillin, that means it has something in its DNA that is now making it ampicillin-resistant.

What factors might influence transformation efficiency? Explain the effect of each factor you mention.

There are numerous factors that could influence transformation efficiency, such as the bacterial medium, growth phase of bacteria, and effectiveness of heat shock. There are certain bacterial mediums that optimize transformation efficiency, Luria Broth, being one of them. If there isn’t enough of a variance between the heat shock, the foreign DNA may not be able to enter the cell, or if there is too much variance the bacteria could die. There are certain growth phases of bacteria. The log phase is when the bacterial population grows rapidly, and is the stage where transformation efficiency is high.

Conclusion: This lab covered the topic of basics transformation in a bacterial cell with topics of plasmids and antibiotics being applied and how transformation takes place, as well as aseptic procedure and how to follow it. We learned by which measures transformation efficiency can be optimized- by adjusting variables such as size of the bacterial colony used, the amount of plasmid used, technique, and incubation times. We can apply this to our project by creating a tutorial that shows how to transform bacteria with maximized transformation efficiency and aseptic procedure.