My research Theme is Screening and Analyses of Error Prone PCR Generated Green Fluorescent Protein (GFP) Variants Cloned in pET-BLUE1

The Significance of my work is :

• GFP is useful for label.
• Target because since GFP fluoresces one can shine light at the cell and wait for the distinctive green fluorescence associated with GFP to appear.
• Using error prone PCR we are creating new variants of the GFP.
• E. Coli is useful because they are relatively easy to work with and because there is a vast amount of previous knowledge about them.

Talking about the Specific Aims they are :

I. New Up Primer design at ATG initiation codon of the GFP gene in pGLO.
II. Amplify the GFP region under normal and mutagenic conditions.
III. Clone the GFP amplified region into the pET-Blue1 Vector.
IV. Analyze GFP variant protein properties.
V. Characterize GFP variants in E. coli.

Using the Specific Aims established for this semester. I can say that the progress we have done can be classified in number 2. That means  we have done lees of a 50% of our main Goals. The specific aims that we have accomplished are : the I .New Up Primer design at ATG initiation codon of the GFP gene in pGLO, and
II. Amplify the GFP region under normal and mutagenic conditions.

This picture taken in October 23rd 2008  shows the results of a PCR under normal and mutagenic conditions

Lane 1 – ladder 100bp
Lane2 – old primer used
Lane 3- New primer at ATG initiation codon of the GFP gene in pGLO.
Lane 4 – Mutagenic PCR round 1
Lane 5 – Mutagenic PCR round 2
Lane 6 – Ladder 1 kbp