The process involves preparing a column and adding an elution buffer along with a slurry or molecular matrix to create a uniform packed matrix. The column is then loaded with GFP extract, and its progress is monitored using UV light.
Heat Shock Temp. C
ice (<0)
water bath (45)
ice (<0)
water bath (37)
Tayte Kozlowski
1. The proteins in the 'Denatured' tube are denatured by adding SDS (sodium dodecyl sulfate). When boiled, SDS breaks up the weak peptide bonds between amino acids in proteins, smoothing them out so they exist in rope-like chains. The strands of protein can then be placed in an SDS PAGE (gel) to be arranged by size. Smaller strands travel faster through the gel than longer strands.
2. Conversely, 50% glycerol is added to the 'Native' tube. Native proteins are not as charged as denatured protein, as they have not been unraveled. These will be ran through the gel to see the difference between denatured and non denatured protein.
3. After the gel was complete, results were analyzed to determine the molecular weight of the sample, verifying its authenticity. The standard molecular weights provided were graphed on semilog graph paper and a standard line was drawn.
4. The migration distance of the denatured protein was measured, and then using the standard line, it was found that the molecular weight of out sample was about 27,000 da. Since the known molecular weight of GFP is also 27,000 da, it can be confirmed that our sample is truly GFP.
1. The column is prepared (mounted with cap on).
2. Elution buffer and slurry, or molecular matrix (a semi-solid substance used to separate proteins), is put into the column and a beaker is placed under the column to collect the buffer as the cap is removed. This creates a uniform packed matrix for the protein-filled supernatant to flow through.
3. The column is loaded with GFP extract and the progress of it in the gel matrix is monitored using a UV light. Gravity pulls the GFP based on size (larger move faster through the column). Once the GFP nearly reaches the bottom of the column, fractions are collected in the microtiter plate. Once the GFP has been completely eluted, 30 micro liters of the brightest elution was added to a tube and labeled 'Native', and 30 micro liters was added to a tube labeled 'Denatured.'
1. Bacteria from the LB/AMP/IPTG (glowing) agar plate is spread over 2 more plates and then incubated overnight for growth.
2. Cell growth that presented the most expression of the two plates is collected and suspended with lysis buffer
3. A cycle of freezing, thawing, and then vortexing (mixing) the tube is completed 3 times, and then the tube is centrifuged. This causes the protein to release from the bacteria, leaving it in the supernatant (on top) contains all of the GFP
4. The supernatant is transferred into a new tube.
1. 5 colonies of E. coli is added to tube labeled -DNA that contains Cacl2 solution and then placed on ice. After a quick mix, 250 micro liters of the -DNA cell suspension is added to a tube labeled +DNA, and pFlouroGreen plasmid is added to the +DNA tube. The neutral CaCl2 compound triggers a chemical transformation, as the heat shock slows the movement of the molecules in the cell and the plasmids easily enter the E. coli bacteria.
2. Recovery broth is added to the tubes and they are left to recover. The broth contains nutrients necessary for growth.
3. The -DNA and +DNA are streaked on agar plates and bacteria growth is monitored. Control plates (-DNA) that don't contain bacteria with the pFlouroGreen plasmid will act as normal DNA, and will not grow in ampicillin. Transformation plates (+DNA) will show growth in ampicillin (because of the AmpR gene) and will glow in IPTG compound, as the IPTG allowes gene expression.
The Process of Making GFP
Phase IV: Analysis of Purity using SDS-PAGE
The goal of phase 4 is to determine how pure the protein is by looking at the size of the protein.
Phase III: Purification (chromatography)
The goal of phase 3 is to isolate the GFP through the use of column chromatography.
Phase II: Inoculation/Isolation of GFP
The goal of phase 2 is to make as much GFP containing bacteria as possible.
Phase I: The Transformation of pFlouroGreen
The goal of phase 1 is to transform the GFP host E.coli bacteria with the pFlouroGreen plasmid.
