From Liquid Research
The goal of this project is to alter the DNA sequence of the GFP gene in pGLO to cause the expressed protein to fluoresce blue or yellow under ultra-violet light.
Lab Head - Madi Taylor
Lab Assistants - Andrew Zola, Jack Hickey, Teddy Leithead
Green Fluorescent Protein (or GFP) is a very commonly used and highly applicable protein in terms of biological research. Its usage began in 1994 with the cloning and successful transformation of GFP from a species of jellyfish known as Aequorea Victoria. Because it is found naturally and is easy to produce and handle, GFP has been very useful in the field of biology. The protein can be used as a marker to easily view and track a point of interest link title. Because of these useful traits , the protein has been studied extensively. Scientists have been able to successfully mutate the GFP gene sequence to make it fluoresce a wide range of colors. Many mutations only require the change of a few base-pairs. Colors including red, blue, yellow, and orange have been successfully produced. We have created a project to use site-directed mutagenesis to mutate GFP into BFP and YFP, which we will then transform and use to make a multi-colored fluorescent drawing. At present, we have successfully created Blue Fluorescent Protein (BFP) by mutating the GFP gene in pGLO. We are currently working on producing the Yellow Fluorescent Protein (YFP).
In order to mutate pGLO into pGLO-B that expresses BFP, we took pGLO and transformed it into competent cells to make our starter plate. We then picked one of the colonies from and grew it in liquid LB Agar and Ampicillin. We miniprepped the cells in order to isolate the pGLO plasmid. We then ran a PCR reaction will be happening with primers containing the necessary mutation to turn the GFP gene sequence into the BFP. Afterwards, we used DPN1 to digest all the parental plasmids, leaving us with the one containing the BFP gene (pGLO-B). Naturally occuring plasmids such as pGLO have periodic Methyl groups attached to them, while the mutated plasmids produced in the PCR reaction do not. DPN1 finds these methyl groups and cuts them, so that the plasmid falls apart; therefore, we are only left with the mutated plasmid. We then transformed pGLO-B into or competent cells. After plating the bacteria on Amp/Ara plates and incubating for 16 hours, we exposed the plates to UV light and the cells fluoresced blue, meaning the site-directed mutagenesis was successful. The process of essay writing will be much easier with MarvelousEssays.Com as there are a lot of highly professional and talented writers who are always eager to help you out with any sort of academic assignments regardless of the complexity levels. I do know what I�m talking about! We are now repeating the process to create YFP, which requires 3 mutations made in three separate PCR reactions with a DNP1 digestion between each.
At present, we have successfully mutated pGLO (containing the GFP gene) into pGLO-B (containing the BFP gene). Successful mutations of t1540c and t1542c on the pGLO plasmid caused the expressed protein to fluoresce blue instead of green. By utilizing Stratagene and PrimerX primer design websites, we were able to design the primers to create the above mutations via a PCR reaction. After the first round of mutagenesis and transformation, we decided to use Stratagene primers for the rest of the project because they produced more reliable results.
Transformed pGLO-B under UV light.
Transformed pGLO and pGLO-B on the same plate, under a UV light.
Because we’ve had a number of failed experiments, we need to continue to try to verify whether or not we still have viable DNA
Up to this point we’ve ran two gels looking for GFP DNA yet have not found any DNA.
We need to perform these experiments to answer why there was DNA in our tube 3 months ago, yet supposedly no DNA now.
1. We’ve performed the gel electrophoresis incorrectly a number of times and therefore are just not getting results
How to test: Have someone else run our DNA in a gel and run a control along with it involving something we know contains DNA
2. An enzyme now exists in the tube that has degraded the DNA so it is no longer usable
How to test: Test for DNA/protein (enzymes) with the nanodrop
3. Was there a problem during PCR digestion?
How to test: Test post-PCR sample to determine if there is DNA in there. If there is no DNA in the post-PCR sample, yet DNA in our tube, could we have over-digested or over-spun our sample?
Week of 1.25.12
Week of 2.6.12
Goal: Take pGLO and transform it too make sure we still have the plasmid.
Made LB plates and LB/Amp/Ara plates. Streaked dh5alpha on two LB plates from two different Master plates Incubated 16 hours overnight
Took out plates in morning and there was no growth. Streaked another plate with dh5alpha and put it in different incubator for 16 hours.
We have been able to mutate the sequence of the GFP gene in pGLO to express BFP, which causes the transformed bacteria to fluoresce blue under ultra-violet light. We are currently working on mutating the GFP gene to YFP (yellow).