Copy of lab 10 GFP purification lab report PDF

Preview

Summary

lab report gfp purification fully done. results included...

Description

Biotechnology Lab # 10 GFP Purification Introduction: The genetic information for the protein GFP, or Green fluorescent protein, is contained in the plasmid pGreen, which is double-stranded DNA. This plasmid came from the Aequorea Victoria, a bioluminescent jellyfish found in the deep waters of North America's west coast. This protein is crucial because it may be used as a visual tag or indication by fusing with other proteins.Only transformed cells can proliferate in the presence of ampicillin due to the released beta-lactamase. In the presence of ampicillin, only transformed bacteria with the pGLO plasmid and the ability to make beta lactamase can survive. GFP is a hydrophobic protein, by using different salt solution buffer we can separate GFP from a mixture of proteins. We have created E-Coli transformants with pGLO plasmid which contain the bla-g , arasi to produce GFP protein. In order to start this experiment there will be three buffers used. In buffer one there will be a high concentration buffer, in the second will be medium salt concentration and the third will be low salt concentration. This experiment starts off with hydrophobic pockets being exposed to and the proteins then bind to matrix.In the second buffer proteins are weakly bound to the column fall off. The strong hydrophobic proteins will remain bound.In the last buffer the hydrophobic pockets of GFP are exposed.

Chromatography is a very effective method for isolating proteins from a complicated mixture. GFP must be isolated from hundreds of endogenous proteins found in bacteria. A cylinder, or column, is tightly packed with minuscule beads in chromatography. These beads create a matrix that proteins must travel through before being gathered. The matrix exhibits "affinity" for the target molecule (GFP), but not for the other bacterial proteins in the mix. GFP adheres to the column, allowing it to be distinguished from bacterial contamination. The isolation and multiplication of a single gene is referred to as cloning. Because a single bacterial colony is derived from a single bacterium, all of the bacteria in the colony are genetically identical and are referred to as clones. Single isolated colonies of both green and white bacteria are chosen for transfer to the culture media after picking colonies (or clones) from their agar plates. Single isolated colonies that are at least 1-2 mm apart from other colonies on the agar are unlikely to be contaminated with other bacteria. Centrifugation is a process that uses high-speed spinning to separate molecules based on their size (similar to the spin cycle in a washing machine, when the clothing is compared against

the washer walls). A single centrifugation operation will be used to separate the heavier bacterial cells from the liquid growth medium in this lab session. Centrifugation produces a "pellet" of bacteria at the tube's bottom, as well as a liquid "supernatant" above the pellet. The collection and concentration of bacteria are the initial steps towards isolating GFP from bacteria cultured on liquid medium.

Methods: PROTOCOL 1. Lesson 2 Growing Cell Culture: Pick Colonies 1. Take the transformation plates out of the incubator and inspect them under a UV light. Identify multiple green colonies on the LB/amp/ara plate that are not contacting other colonies. On the LB/amp plate, look for many white colonies. 2. Collect two culture tubes with the LB/amp/ara growth medium. One should be labeled "(+)" and the other "(-)". Gently contact a green colony with a sterile loop before immersing it in the "(+)" tube. Rep with a fresh sterile loop for a white colony and place it in the "(-)" tube (it is critical to select just one colony). To spread the entire colony, spin the loop between your index and thumb. 3. Cover the tubes and cultivate for 24 hours at 32°C or 2 days at room temperature in a shaking incubator, shaking water bath, tube roller, or rocker. When feasible, remove the item and shake it with your hand. Protocol 2 Lesson 3 Purification Phase 1: Bacterial Concentration

1. Write your name and class period on one microcentrifuge tube marked "(+)." Take your liquid cultures out of the mixer and examine them under UV light. Any color discrepancies between the two cultures should be noted. Transfer 2 ml of "(+)" liquid culture into the "(+)" microcentrifuge tube using a fresh pipet. In the centrifuge, spin the microcentrifuge tube at full speed for 5 minutes. The pipet used in this phase can be washed in a beaker of water many times and reused for the rest of the laboratory session. 2.Strain the supernatant from the particle and examine it under UV light. 3. Fill the tube with 250 l of TE buffer using a rinsed pipet. By rapidly pipetting up and down multiple times, the particle is fully resuspended. 4.To begin enzymatic breakdown of the bacterial cell wall, add 1 drop of lysozyme to the resuspended bacterial pellet with a washed pipet. Lysozyme is an enzyme that cleaves polysaccharide (sugar) residues in the bacterial cell wall to destroy (or lyse) the cell wall. By gently flipping the tube, the contents will be mixed. Under UV light, examine the tube. 5. Place the microcentrifuge tube in the freezer until the next time you need it in the lab. The bacteria will totally burst as a result of the freezing. Protocol 3 Lesson 4 Purification Phase 2: Bacterial Lysis 1. Take the microcentrifuge tube out of the freezer and defrost it with your hands. Place the tube in the centrifuge and spin for 10 minutes at full speed to pellet the insoluble bacterial debris. 2. Prepare the chromatography column while your tube is spinning. Remove the lid from the prefilled HIC column and snap off the bottom. Allow 3-5 minutes for the liquid buffer to drain completely from the column.

3. Prepare the column by pouring 2 mL of Equilibration Buffer into the column's top. This is accomplished by using a rinsed pipet to add two 1 ml aliquots. Drain the buffer until it reaches the 1 ml mark on the column. Cap the top and bottom of the column and keep it at room temperature until the next laboratory period. 4. Remove your tube from the centrifuge after 10 minutes of spinning. Use the UV light to examine the tube. Transfer 250 l of the "(+)" supernatant into a fresh microcentrifuge tube labeled "(+)" using a new pipet. Rinse the pipet thoroughly for the remainder of this lab period's instructions. 5. Transfer 250 l of binding buffer to the "(+)" supernatant using a well-rinsed pipet. Keep the tube refrigerated until the following laboratory session. PROTOCOL 4. Lesson 5 Purification Phase 3: Protein Chromatography 1. Number three collection tubes 1-3 and place them on the foam rack or a rack provided by your instructor. Remove the top and bottom caps from the column, then insert it in collecting tube 1. Proceed to the following step below after the last of the buffer has reached the HIC matrix's surface. 2. Carefully and gently load 250 l of the "(+)" supernatant onto the top of the column with a fresh pipet. Allow the supernatant to trickle down the side of the column wall by pressing the pipet tip against the column wall slightly above the upper surface of the matrix. Using a UV light, examine the column. Make a list of your observations. Transfer the column to collecting tube 2 after it has stopped pouring.

3. Add 250 l of wash buffer to the rinsed pipet and let the full volume run into the column. Use the UV light to examine the column. Make a list of your observations. Transfer the column to tube 3 after it has stopped leaking. 4. Add 750 l of TE Buffer to the washed pipet and let the full volume run into the column. Use the UV light to examine the column. Make a list of your observations. 5. Examine each of the three collecting tubes for color changes. Wrap the tubes in cling film or plastic wrap and keep them in the refrigerator until the next lab time.

Results:

Blue line= Hydrophobic beads above

Discussion: GFP's unique properties make it possible to purify it from bacterial cell proteins using HIC columns. The HIC matrix preferentially binds hydrophobic GFP molecules while allowing

bacterial proteins to flow through the column when put in a buffer with a high concentration of salt. The wash buffer began to enable the hydrophobic bacterial proteins to move through the column while binding to the GFP proteins in the first tube. The remaining bacterial proteins, as well as a few GFP proteins, are washed out of the column in the second step.Tube three demonstrates that the purified GFP proteins are completely eluted into tube three.

Bibliography: -“Home.” Biotility, https://biotility.research.ufl.edu/lab-experiences/gfp-lab/....