Safety Considerations. Notes for the last TWO exercises

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1 Notes for the last TWO exercises! The notes that follow outline the activities for the last TWO exercises: 1- Molecular Biology (blue on your Calendar), and 2- Protein Expression (red on your Calendar). You will be expected to construct and write out each individual procedure in your notebook before you conduct the experiment. You can do the prelab. write-ups for each day on the day itself (no need to write up the entire exercise for the first day). Be thorough, complete, plan well, and run all expected controls. Do not throw out any material until the exercise is over. 1 Safety Considerations! Observe all standard safety practices in the lab.! Dispose all waste properly. If you are not sure, please ask.! Maintain sterile technique throughout, for your protections and for the protection of your reagents and strains.! 1-nitropyrene, our mock substrate, is a potential carcinogen. It is one of the chemicals present in diesel fuel exhaust that makes us nervous about breathing diesel fuel exhaust. Minimize your exposure to this compound and dispose of it safely. PDF and related info (posted).

2 Overview of last 2 exercises: 1- Transformation & Expression, 2- Bioassay and carrying cost. Objective 1: transform plasmid into cells and test for functional expression of cloned gene. Group A: 2 & 4 April Group B: 9 & 11 April Tues: Transformation and Selection. Thurs: Count transformants, restreak onto a plate containing IPTG. Objective 2: determine whether the cloned gene is functional and determine the cost of carrying the plasmid and expressing the gene. Group A: 9 & 11 April Group B: 16 & 18 April Tues: first section will use transformed bacteria to innoculate liquid cultures, and streak plates containing mock substrate. Second section will plate cells from liquid cultures, and streak plates containing mock substrate. Thurs: compare colony counts to assess metabolic costs of carrying plasmid and expressing gene. Look for signs that transformed bacteria metabolized mock substrate. Molecular biology: Transformation with Plasmid, Gene Expression Objective: transform cells with the engineered plasmid, check for expression of a gene on the plasmid. Group A: 2 & 4 April Group B: 9 & 11 April Tues: Treat competent cells with engineered plasmid. Select transformed cells based on ability to tolerate the antibiotic ampicillin (resistance is encoded by a gene on the plasmid). The transformation will also be performed on a positive control: a plasmid that has not been subjected to digestion and ligation etc. Thurs: Count transformed cells, re-streak onto a plate containing IPTG in order to learn whether cells can express the plasmid-borne cloned gene. For this we will exploit the fact that our control plasmid carries the gene for green fluorescent protein. Friday or later: view plates, count fraction of cells expressing GFP. Also re-view your initial selection plates. In your report, estimate of the number of cells transformed with each of the engineered plasmid and the control. Note the fraction of transformed cells that express GFP from the control plasmid. Discuss efficiency.

3 Protein Expression: Bioassay and Carrying Cost Objective: test for functional expression of cloned gene (evidence that the bacteria have a new biological activity, i.e. phenotypic transformation). Assess the biological costs that attend possession of extra DNA and production of extra proteins. Group A: 9 & 11 April Group B: 16 & 18 April Tues: Streak cells transformed with engineered plasmid onto a plate containing IPTG and a mock substrate. Use cells transformed with engineered plasmid to innoculate four liquid cultures (3 ml each but different additives). Streak LB plate and LB +Amp plates with 25 ul cells from each culture. Thurs: Examine plates for signs that the mock substrate has been metabolized. Count colonies from each liquid culture on each plate to assess cost of carrying the plasmid, and expressing the gene. Relevant Book Sections We will follow the protocols provided in various industry-standard kits, instead of the protocols described in these chapters, but the chapters provide good background and explanations.! Transformation: exercise 20! Expression from a T7 promotor: exercise 22! DNA and genetic elements: pgs Likewise, protocols can be developed from our Molecular Cloning Manuals. Maniatis is a staple in the field, there are now even journals and video journals devoted to these techniques and the tricks embedded in them. 6

4 Day 1: Molecular Biology execution plan! Get the transformation started.! Pour plates of LB agar containing ampicillin (100 ug/ml). The agar will be provided in molten form (55 C, hot). Students will have a chance to try pouring. Ampicillin (Amp) should be added and agar should be poured when it is still molten but cool enough to touch. (Note that our control plasmid encodes resistance to kanamycin (Kn) instead of Amp, so plates to select for the outcome of the control transformation will be LB-Kn instead of LB-Amp).! We will be adding Amp (Kn) from a stock solution that is 100 mg/ml, into 500 ml batches of molten agar. Calculate how much Amp to add.! Complete the transformation as per attached protocol.! Plate out cells on the LB-Amp and LB-Kn plates (cartoons follow). 7 Day 1 theory: Transformation Transformation means causing a bacterial cell line to take up foreign DNA. The term refers to the original observation that bacteria acquired new biological properties upon acquiring foreign DNA: they were transformed into a new strain more like the one from which the DNA was derived. For an intact plasmid with no toxic genes (puc or pbr322), 10 8 colonies are obtained per µg DNA used. (For a 4 kbp plasmid, at 600 g/mole bp, the MW is 2.4 x 10 6 g/mole = 2.4 x µg/mole. 1 µg = 1 µg * (6 x molec/mole) / (2.4 x µg/ mole) 2.5 *10 11 molecules of plasmid colonies means we got 10 8 cells transformed. The transformation efficiency is therefore 1 cell / 2500 molecules of plasmid. Transformation will be less efficient when the plasmid is large or has incomplete ligation. It will not occur if a plasmid is not stable (it is not circular or does not contain an origin of replication). Our engineered plasmid should have a replication origin, but it will also likely retain nicks not sealed by ligase. Therefore, we can expect LOWER efficiency. 8

5 Molecular Biology: Protocols used Transformation and Selection We will transform our plasmids directly into an expression strain of E. coli (T7 express) following the protocol that follows. T7 express has that name because it carries the gene for T7 RNA polymerase in its chromosome. Thus it can produce the RNA polymerase that would normally be produced by T7 virus. Genes behind a T7 promoter will be transcribed by this polymerase, and expressed. Hence the name of the cell line. In order to get cells to take up foreign DNA we have to weaken their cell walls and permeabilize them. The resulting cells are called competent cells because they are competent to take up DNA. However they are fragile and must be handled with some care. Since only a few cells will actually take up the DNA and copy it (see efficiency, above), we need a good selective screen to identify the cells that do. Usually we choose a screen that kills all cells that are not transformed (zero background). Since a gene conferring ability to degrade ampicillin is on the plasmid, only cells with the plasmid might be expected to survive. (However, look carefully at your plates after they have been allowed to grow for several days.) 9 Transformation: introduction of foreign DNA into cells! Cells have natural controls on access of foreign DNA.! We will use cells whose membranes have been permeabilized, by chemical treatment (CaCl2, DMSO and more).! We will also heat-shock them.! DNA aggregated to exterior phospholipid head groups with Ca 2+ can be brought in.! Once the DNA gets in, it can be degraded, integrated, or independently replicated (requires a replication origin ). After-the-fact elimination of foreign DNA lowers the efficiency of transformation further.! We will transform with our engineered plasmid in parallel with a control plasmid that has not been subject to endonuclease digestion etc. The positive control will be a plasmid carrying the green fluorescence protein (GFP) gene. (I.e. each student will run a pair of transformations in parallel.) 10

6 Transformation T7 Express Iq Competent E.coli (High Efficiency) High Efficiency Transformation Protocol (C3016) Sterile Technique Hygiene for you, and hygiene for your bacteria. Plating out bacteria. Overview For C3016H, perform steps 1-7 in the tube provided. 1. C3016H: Thaw a tube of T7 Express I q Competent E. coli cells on ice for 10 minutes. C3016I: Thaw a tube of T7 Express I q Competent E. coli cells on ice until the last ice crystals disappear. Mix gently and carefully pipette 50 µl of cells into a transformation tube on ice. 2. Add 1-5 µl containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex. 3. Place the mixture on ice for 30 minutes. Do not mix. 4. Heat shock at exactly 42 C for exactly 30 seconds. Do not mix. ** 5. Place on ice for 5 minutes. Do not mix. 6. Pipette 950 µl of room temperature SOC into the mixture. 7. Place at 37 C for 60 minutes. Shake vigorously (250 rpm) or rotate. 8. Warm selection plates to 37 C. 9. Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in SOC. *** 10. Spread µl of each dilution onto a selection plate and incubate overnight at 37 C. Alternatively, incubate at 30 C for hours or at 25 C for 48 hours. * * Heat shock for 45 seconds (not 30) ** Add 800 µl SOC (not 950). *** We will plate all the cells. First plate 100 µl of cells. Then spin down remaining cells, decant all but 50 µl of supernatant, resuspend cells and plate those. Selection will be accomplished by plating on agar containing 100 µg/ml ampicillin or kanamycin (engineered or control plasmid, respectively). 11 Swab the bench and your hands with 70% ethanol (wear gloves). Move only the items you need into your sterile field, light a burner to direct air currents up, work efficiently keeping tubes and plates covered whenever possible. At the end, extinguish flame, swab down area again including your hands.

7 Day 2 of Molecular Biology: Expression. Execution plan:! Count colonies obtained from transformation using engineered plasmid, vs. control transformation. Also look at the T.A. s negative control transformation (no plasmid). Interpret these results in your report.! Streak colonies of the positive control transformation onto LB agar that contains Kn + IPTG.! For each colony you streak onto the LB Kn + IPTG plate also streak with the same colony onto an LB Kn plate, and number the streaks so you know that both come from the same cell. Question: Why might you want a replicate culture that is not subjected to IPTG? (hint: think ahead to the topic of the next exercise). 13 Day 2 of Molecular Biology: Expression. We will streak cells from a couple of colonies onto plates containing Kn + IPTG. The T.A. will provide plates with negative control bacteria (not carrying a GFP plasmid). Question: What would be an ideal control? c3 3 1 LB Kn + IPTG c1 c2 2 Use a sharpie to mark a plate into six sectors and mark each sector 1 to 3 and c1 to c3 (test colonies 1 to 3 and control colonies 1 to 3). Write on the bottom of the plate, not the lid. Streak into each sector after picking up cells from one colony (a different colony for each sector). Don t pick up all the cells from your chosen colony, because you need to pick up more for your second plate. 14 c3 LB Kn 3 1 c1 c2 2

8 Day 2 of Molecular Biology: Expression. Theory. Only cells carrying the plasmid will grow on Amp. However which of these plasmids contains the gene of interest? In order for T7 express cells to express genes cloned behind the T7 promotor, we must turn on production of T7 RNA polymerase. This is the only polymerase that will recognize the T7 promotor. (We should get very selective expression). To turn on (induce) expression of T7 RNA polymerase we will streak transformed cells onto an LB plate containing IPTG (provided). Our positive control transformation should provide a very visual measure of whether the plasmidborne gene is expressed because in that case the gene is for green fluorescent protein (GFP). We will be able to tell if it is expressed by illuminating the colonies with a black light. We will check for expression either by appointment on Friday or on the following Tuesday. 15 Protein Expression: Bioassay and carrying cost. Objective: determine whether the cloned gene is functional and determine the cost of carrying the plasmid and expressing the gene. Group A: 9 & 11 April Group B: 16 & 18 April Tues: first section will use transformed bacteria to innoculate liquid cultures, and also streak plates containing mock substrate. Second section will plate cells from liquid cultures onto different plates, and also streak plates containing mock substrate. Thurs: count colonies from each liquid culture on each of the two plates. Look for signs that transformed bacteria metabolized mock substrate. In your report: discuss the metabolic costs of expressing a gene, as well as the cost of carrying the plasmid. 16

9 Carrying cost day 1: Execution Plan for Section 1 Transfer the cells of three colonies transformed with engineered plasmid into 400 µl of LB liquid in a sterile eppendorf tube, mix completely to make homogenous suspension. From the 400 µl, transfer 100 µl to 0.8 ml of LB medium in sterile eppendorf. transfer 100 µl to 0.8 ml of LB + Amp medium. transfer 100 µl to 0.8 ml of LB + IPTG medium. transfer 100 µl to 0.8 ml of LB + Amp + IPTG medium. Shake at 37 C for two hours. Question: In which culture do you expect the largest fraction of cells to retain the plasmid? Rank the cultures from the one that should retain the most plasmidcarrying cells (as fraction of total) to the one that should retain the least. Carrying cost day 1: Execution Plan for Section 2 Obtain an LB plate and an LB Amp plate. Mark each with 4 sectors, writing on the bottom of the plate. Label the sectors as below, writing on the bottom of the plate. Centrifuge cells out of liquid cultures, decant most of supernatant leaving 50 µl and resuspend cells in that. Use 25 µl of the cells and spread them on the appropriate sector, for each of your plates (25 µl on ea). Incubate the plates at 37 C overnight. (T.A. will then refrigerate plates.) LB only LB Amp LB IPTG LB IPTG Amp LB medium LB only LB Amp LB IPTG LB IPTG Amp 17 from LB from LB IPTG from LB Amp LB from LB Amp IPTG from LB from LB IPTG from LB Amp LB-Amp from LB Amp IPTG18

10 Carrying cost: theory! Maintenance of the plasmid requires ongoing metabolic commitment.! When ampicillin is present, the commitment is required.! When IPTG is present, the cost of carrying the plasmid is augmented by the cost of expressing the gene.! When drug is not present, bacteria bearing plasmid have a growth disadvantage, due to the cost of carrying the plasmid. Thus in a liquid culture where they compete directly with all other bacteria, the plasmidbearing bacteria will tend to divide less frequently and dwindle as a fraction of the total population.! This disadvantage is accentuated when IPTG is present because now the plasmid-carrying bacteria are also expressing a gene. They can be expected to dwindle even faster unless the gene they are expressing improves their ability to compete.! To learn what fraction of the total population is represented by a subset of bacteria, we plate 25 µl of liquid culture on LB agar where all bacteria will produce colonies, and also 25 µl on LB + Amp agar where only plasmid-carrying bacteria will produce colonies. The ratio of the colonies on the LB+Amp plate / LB plate gives the fraction of colonies with the plasmid.! We do this for each type of liquid culture to assess the relative costs of carrying the plasmid vs. expressing the gene. 19 Bioassay Day 1: Execution plan.! Streak colonies from the transformation with the engineered plasmid onto LB plates containing Amp and IPTG and 1-nitropyrene ( mock substrate, S). For each colony you streak onto the Amp, IPTG + S plate also streak with the same colony onto an LB Amp + S plate, and number the streaks so you know that both come from the same cell. c3 3 1 c1 c2 LB Amp + IPTG + S also LB Amp + S 2 Use a sharpie to mark a plate into six sectors and label them, writing directly on the bottom of the plate. Streak into each sector after picking up cells from one colony (a different colony for each sector). The C sectors will be streaked with control colonies (from the GFP transformation) that should NOT be able to degrade substrate. The experiment sectors will be streaked with colonies bearing the engineered plasmid. 20

11 Bioassay day 1: Theory. To screen for functional expression of our cloned gene for herbicide degradation, we will streak cells from a couple of colonies onto plates containing Amp, IPTG and a coloured compound that resembles the herbicide dinoseb ( S for substrate). Any colonies able to degrade the 1-nitropyrene will cause a colour change in the agar. This will indicate successful cloning of a functional gene. We will use cells transformed with the GFP-bearing plasmid as negative controls. Because expression of the gene can be a severe burden, especially if the gene produces toxic product, or metabolism of 1-nitropyrene produces toxic product, we want a control plate which will allow us to interpret instances in which the cells may fail to grow on the substrate-containing plate. The control we will use is to plate on LB Amp with substrate but no IPTG. Question: what is another control we could use? Question: what could we expect to learn from that control? Bioassay day 1: Theory. Expression of the gene will be induced by IPTG at 1 mg/ml in the LB agar (along with 50 µg/ml ampicillin). We will incorporate the proxy 1-nitropyrene into the LB agar. Nitropyrene is yellow in colour. Upon metabolism the yellow colour disappears. 1-nitropyrene will be present at 16 µg / ml in the agar. 1-Nitropyrene Dinoseb Rafil et al (1991) Appl Environ Micro pg FIG. 1. Clearance of 1-nitropyrene around colonies of bacteria that had been isolated from human intestinal microflora and showed nitroreductase activity. 22

12 Bioassay day 2! Carrying cost: count colonies in each sector of each of the two plates. Tabulate the results.! Bioassay: examine the experimental (1-nitropyrenecontaining) plate and look for distinctions between effects of bacteria carrying the engineered plasmid vs. effects of bacteria carrying the control plasmid. Pay attention to reproducibility of the effect (eg. between progeny of your three different experimental colonies.) Review: Induction of the T7 RNA polymerase gene on the T7 express, chromosome... In E coli T7-express cells, the lac operator controls expression of the gene for T7 RNA polymerase, instead of lactose metabolism. LacI+IPTG RNA polymerase mrna Ribosome 23 Operator Promotor DNA encoding gene for T7 RNA polymerase. 24

13 Review: Now back to the plasmid:expression of the gene that follows the T7 promotor. Protein encoded by plasmid-born gene. mrna T7 RNA polymerase* Promotor recognized exclusively by T7 RNA polymerase (*structure shown is actually that of NS5B polymerase) 25