The Experiment. Protocols used. 1- Site-directed mutation. 2- Transformation and Selection. 3- Phenotypic screen. 4- Carrying cost

Transcription

1 The Experiment You will be expected to construct and write out each individual procedure in your notebook before you conduct the experiment. You can do this on a day-today basis. Be thorough, complete, plan well, and run all expected controls. Do not throw out any material until the exercise is over.! We will not be following the protocols from the book. However, the book is a good source of background information.! As is standard for molecular biology experiments, all protocols will be obtained from the companies we buy the material from. In this area technology is changing very rapidly so textbooks have difficulty keeping up. Feel free to visit the companies web sites for additional information.! The QuickChange manual has been posted on the web site so you can read about it. Protocols used 1- Site-directed mutation QuikChange II Site-Directed Mutagenesis instruction manual. Instead of 50 µl reactions as on page 7 of the manual, we will conserve reagents by using 25 µl reactions. 2- Transformation and Selection We will transform our plasmids directly into an expression strain (T7 express) following the protocol that follows, and select based on ability to grow on kanamycin. 3- Phenotypic screen We will score the fractions of plasmids that carry the desired mutation based on the protocol that follows. Since the mutations are expected to alter the colour of protein fluorescence, we can use a visual screen under UV light. 4- Carrying cost The T.A. will prepare replicate liquid cultures wherein otherwise identical cultures will have either -1- no additives, -2- added Kn, -3- added IPTG or -4- added IPTG and Kn. These will grow for 6-8** hours in advance of class. In class you will plate out **30 ul of the culture on each of an LB/ Kn plate or an LB plate, for each of the cultures. Two days later we will count the number of colonies obtained in each case to learn what fraction of the bacteria stopped carrying the plasmid, and how that was affected by whether the plasmid came with a burden of protein expression and/or defense against the drug. 35

2 Protocol Day 1: QuikChange Reaction Master mix Sterilized water 10X reaction buffer Template DNA (40ng/ul) 10mM dntp pfu DNA polymerase(2.5u/ul) 19.5ul 2.5ul 0.5ul 0.5ul 0.5ul. Add 0.75 μl of 10 μm primers**. (Different people use different primers, see next page.) Tubes will be placed in the Thermal Cycler (used for the PCR experiment) and allowed to run into the night. The T.A. will retrieve the samples and store them in the -80 C freezer until the next class. Thermal-cycle 95 2min 16 cycles of: { 95 30sec 55 1min 68 5min } 68 4min Digestion of parental DNA** Bring DNA to 37 C in a heat block. Add 0.5 ul DpnI (10U/μl) to the product of PCR directly. Incubate at 37 C for 1 hr. **This will be done by the T.A. However please write this up in your prelab notes as if you would do it, but with the notation that the following steps will be executed by Ting Wang. Section 1 Section 2 Primer to use Name Locker Group Primers Danielle Edwards L41C An Lien Ho L35C Joanna Ng L35C Niranjana Warrier L44C Kaeto Vin-Nnajiofor L34C Morgan Sizemore L34C Corey Lee L31C Matthew Wolfe L31C Derrick Lewis L28C Travis Johnson L38C Jessime Kirk L38C Carl Archemetre L44C Falak Patel L35C Robert Reed L41C Dylan Woolum L35C Kalen Wright L41C Hagen Smith L38C A.-F. Miller 2013

3 Protocol: Day 2: transformation into bacteria Transformation of bacteria Gently thaw competent cells on ice. Label one tube mutant and the other WT (wild-type: not mutated), and also include your name/initials. Add 1 µl of the QuikChange reaction mixture to the tube labeled mut and swirl gently to mix. For the WT control, add 0.3 µl of parental plasmid DNA to a second 50 µl aliquot of cells and swirl gently to mix. Parental DNA (i.e. the parent that gave rise to the mutant) will be provided by the T.A. this is just a little bit more of the same DNA we used as the starting point for our quickchange reaction. Incubate both tubes 30 min on ice. Transfer to a heat block at 42 C for 10 seconds. Return to ice for 5 min.! We will assess different transformed colonies on the basis of their phenotype to learn what fraction of the plasmid that transformed the cells carried the desired mutation.! Obtain two LB/Kn plates. Spread 100 µl of IPTG stock solution on ONE of the plates and label that one LB/Kn,IPTG.! Divide up the area in each plate into six sectors as per the drawing below, left.! From a plate of your bacteria transformed with mutated plasmid, locate three colonies that are isolated. On the bottom of the petry dish circle and number each one (see below, right). These are colonies, and. From your positive control LB/Kn plate, locate and label three unmutated (= wild type = WT) colonies. These are colonies,,. Add ml of SOC growth medium to each tube, mix gently. Grow for 45 min. at 37 C. (Note difference from official protocol.) Spin down cells and resuspend in 100 µl SOC. Spread the QuikChange transformation on an LB Kn plate. Spread 10 µl of the +ve control transformation on an LB Kn plate. Also spread 10 µl of the +ve control transformation on an LB plate. Incubate overnight at 37 C. Questions to answer in your pre-lab: Why is Kn (kanamycin) not included in the SOC growth medium? What do we learn by comparing the number of colonies on the LB/Kn plate with the number of colonies on the LB plate? What is the positive control for our transformation? Design a negative control for the transformation. Would you plate that on LB or LB/Kn?

4 Kn! From each of your labelled colonies, lightly touch the colony, taking up 1/3 the cells, and streak these on one sector of a LB/Kn plate: a plate in which the medium contains IPTG as well as Kn. (below, left).! Use a second third of the cells in each of your labelled colonies to streak sectors of an LB/Kn,IPTG plate (below, right). Kn-IPTG Carrying Cost.! The T.A. has grown four liquid cultures of bacteria carrying the GFP-bearing plasmid for 6** hours. She has delivered 0.5** ml aliquots into sterile eppendorf tubes. Take one of each.! One culture medium is just LB another is LB/Kn another is LB/IPTG a fourth is LB/Kn/IPTG! Take four LB plates and four LB/Kn plates. For each type of plate, each plate will be used for a different culture of cell. Label them accordingly (LB, LB/Kn, LB/IPTG and LB/Kn/IPTG)! For each of the four cultures, deliver 30** ul of the cells onto the correct plate and spread them evenly over the entire plate. I.e. deliver 30 ul of cells from the LB/Kn growth on an LB plate labelled LB/Kn. Also on an LB/Kn plate labeled LB/Kn. etc. ** volumes and times still being optimized.! The plates will grow overnight at 37 C. The next day, your TA will move them to the refrigerator. LB growth : LB/Kn LB/Kn growth : LB/Kn 40! You should have 8 plates at the end.! The plates will grow overnight at 37 C. The next day, your TA will move them to the refrigerator. 41

5 Protocol: Day 4:! Compare the intensities of fluorescence of cultures on the LB/Kn/IPTG plate with those on the LB/Kn plate.! Estimate how much more GFP is being produced by cells exposed to IPTG.! Compare the color of your mut bacteria with the color of your WT bacteria.! Did you succeed in mutating the gene?! Which primer did you have? (i.e. was it the Y66H or the T203Y?) Protocol: Day 4: Carrying cost.! Count the number of colonies on each of the plates.! Comparing the results for cells from the same liquid culture, what do you learn by comparing the number of colonies on the LB/Kn plate with the number on the LB plate?! What is the effect of having IPTG in the liquid culture?! What is the effect of having Kn in the liquid culture? Kn Kn-IPTG LB LB/Kn 42 43