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Two new traits the bacteria acquired when they were transformed w/ pGLO plasmid 1) the ability to fluoresce green light when exposed to UV light (due to the GFP gene under the control of the araC regulator 2) ampicillin resistance (due to the beta-lactamase ("bla") gene) If someone gave you a vial of lab stock E.Coli, how would you be able to tell if they were ampicillin resistant? What steps would you take to determine this? You can't tell if bacteria are ampicillin-resistant just by looking at them. You would need to take some of the bacteria and streak them on an LB/amp plate. If the bacteria are viable (able to grow) on the LB/amp plate, then they are resistant to ampicillin. If no bacterial colonies survive, then they are not ampicillin resistant. You're transforming bacterial cells with a plasmid containing gene that allows the bacteria to digest toxic heavy metals. In the same plasmid, you also insert the beta-lactamase gene for ampicillin resistance. a) It's the heavy metal detoxification gene that you're interested in. So, what is the purpose of having the beta-lactamase gene on this plasmid? The ampicillin resistance gene is to allow for selection of transformants (by spreading the bacterial cells on LB/ amp plates after transformation--only those bacterial cells that were transformed with the plasmid will grow on LB/amp plates). To calculate total amount of DNA used (micrograms) the formula is concentration of DNA (microgram/microL) x volume of DNA in microL To calculate fraction of DNA spread on plate the formula is vol. spread on LB/amp/ara plate/ tot.vol. in transformation tube To calculate plasmid DNA spread (microgram) on plate the formula is tot. amt. DNA used (microgram) x fraction of DNA on plate To calculate the transformation efficiency the formula is tot. # of cells growing on LB/amp/ara plate/ amt. of DNA spread on LB/amp/ara plate List the steps of miniprep protocol 1) lysis 2) neutralization 3) binding 4) elution the lysis step in miniprep protocol is when buffer P2 is added to lyse the bacterial cells. This means that the cells are broken open to release all the cellular molecules. Buffer P2 contains detergent (SDS) to break open the cells, and a strong base (NaOH) to denature proteins. the neutralization step in miniprep protocol is when buffer N3 contains an acidic, high salt solution. The acid neutralizes the strong base from the lysis solution, and the high salt causes the chromosomal DNA, denatured proteins, and cellular debris to precipitate. the binding step in miniprep protocol is when after chromosomal DNA, denatured proteins, and cellular debris are precipitated out, the solution containing the plasmid DNA is poured over the column, allowing the plasmid DNA to bind to the column the elution step in miniprep protocol is when the plasmid DNA is released from the column by adding a small volume of low salt or no-salt liquid to the column. When the liquid is spun through the column in the centrifuge, the solution of plasmid DNA will then be captured in the collection tube Why was it necessar to add both arabinose and ampicillin to the LB nutrient growth medium in which the pGLO-transformed cells were inoculated? -Arabinose was necessary to turn on the araC operon and induce expression of the GFP. Without it, bacterial cells would have grown, but they wouldn't express GFP or fluoresce green under UV light. -Ampicillin was added to make sure only bacterial transformed w/ the pGLO plasmid would grow. Without ampicillin, the media could be contaminated w/other, non-transformed bacteria. When you examined the 2 bacterial cultures (one from the LB/amp/ara plate and one from the LB/amp plate) under the UV light, why were both cultures glowing green even though the bacterial colonies were not green on both plates? the white colony (from the LB/amp/ara plate) fluoresces green because the arabinose in the liquid culture media turns on expression of the GFP gene which was previously off on the LB/amp plate (which lacks arabinose), resulting in a green culture. The green colony fluoresces green because arabinose in the liquid culture media continues to induce expression of the GFP gene, resulting in a green culture To centrifuge in the GFP purification procedure means to allow the harvest of bacterial cells containing GFP from the liquid culture (the culture is spun in the centrifuge, and the bacterial cells are pelleted at the bottom of the microcentrifuge tube) To lysozyme in the GFP purification procedure means to break down the bacterial cell walls, beginning the process of lysing the cells (breaking them open to release the GFP) To use the freezer in the GFP purification procedure makes the liquid inside the bacterial cells to expand, causing the cells to break open--completes the process of cell lysis List 3 steps used in the GFP purification procedure 1) centrifuge 2) lysozyme 3) freezer Why was in important to know the lambda max for the Bradford reagent + protein solution? In order for the spectrophotometer to best be able to detect the presence of a molecule (such as a protein), it must be set at the lambda max. Now that the lambda max for the Bradford + BSA protein solution has been determined at ~595nm, we set the spec for 595nm to determine the concentration of BSA protein in solution in activity 11c Xanthophyll is the yellow pigment in lemons. What value would you expect the lamda min to be for a xanthphylls solution. Why did you choose this value? The expected value for the lambda min of xanthophylls would be ~570 nm, the wavelength for yellow light. Since the xanthophylls pigment appears yellow, this means that yellow light is "least" absorbed by this pigment. If the purity of DNA is significantly below 1.8, then it is likely that the DNA plasmid solution has a significant amount of contamination with protein If the purity of DNA is about 1.8, then it's fairly pure If the purity of DNA is significantly above 1.8, then RNA contamination is suspected why use chromatography? to purify a single recombinant protein of interest from over 4,000 naturally occuring E. coli gene products Column chromatography is used for protein purification via hydrophobic interaction GFP is a very hydrophobic protein In column chromatography, GFP which is a hydrophobic protein binds to the hydrophobic beads on the column matrix in the presence of high salt, because w/ out high salt, the hydrophobic parts of the protein will be buried in the core of the protein again, so the protein will no longer bind to the column hydrophobic molecules like hydrophobic molecules and the result is hydrophobic protein sticking together HIC stands for hydrophobic interation chromatography (HIC) the reason hydrophobic proteins will stick to HIC is because of the hydrophobic beads. Hydrophobic molecules are attracted to hydrophobic molecules and stick together more. When a high salt buffer for is added the hydrophobic parts of the proteins become more exposed on the surface of the protein which aids in binding What are the 3 steps with (HIC) Hydrophobic Interaction Chromatography: 1) Bind 2) Wash 3) Elute In HIC, in the bind stage we add bacterial lysate to column matrix in "high salt buffer" In HIC, in the wash stage we wash less hydrophobic proteins (less hydrophobic E. coli protiens) from column in "medium salt buffer". GFP remains bound to column. In HIC, in the elute stage we remove (elute) GFP (which changes shape and no longer binds to HIC) from column with "very low salt buffer" and flows into collection tube Lysis (to lyse) means to break open. We did this to purify the GFP protein by breaking open the bacterial cells to release all the cellular contents (including proteins). Lysozyme is an enzyme that breaks down bacterial cell walls by digesting a polysaccharide in the cell wall. Lysozyme is naturally found in human tears, acting as a bacterial agent to prevent eye infections. The freezing step in GFP purification is important to complete lysis of the bacteria, because freezing will make the liquid inside the bacterial cells expand, causing the cells to explode and break open completely Bradford reagent is a protein indicator Bradford reagent is used to visualize protein molecules in solution the lambda max is the wavelength of light with maximum absorbance proteins like other molecules, interact with light waves and absorb or transmit light energy of various wavelengths the more proteins in solution, the greater the absorbance should be protein molecules are colorless, but when absorbed in solution, they absorb light only in the UV range (specifically at ~200 and at 280 nm) measuring protein concentration in the UV range can be complicated for a # of reasons, including issues of consistency and accuracy the bradford assay is more commonly used to provide better accuracy and consistency for the bradford protein assay the bradford assay uses a colored indicator the bradford reagent reacts proportionally w/ the protein to give a blue molecule BSA stands for bovine serum albumin for lab 11c we used the bradford assay to determine 2 unknown BSA protein solutions protein concentration is usually measured in mg/mL or micrograms/mL When it comes to the standard curve of protein concentration versus absorbance, the best fit line is used to detemine the concentration of unknown protein samples a miniprep is a small-scale DNA plasmid prepartion A DNA plasmid prep ("Mini-prep") is essentially the opposite of bacterial transformation, because we isolated the pGLO plasmid back out of the E. coli cell cultures DNA "preps" are preparations of DNA, frequently plasmid DNA, from transformed cells. DNA plasmid preps are an important way of generating DNA plasmid stocks for long-term storage to be used in future transformations and other techniques