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May Crash Course in Synthetic Biology In order to get some serious training under our belt, do something that helps iGEM, and start work that can be credited towards our project we will be testing Biobrick promoter strength on May 28-June 4th. Those performing the training, make sure that you are familiar with "Measuring the activity of Biobrick Promoters Using an In vivo Reference Standard" by Jason Kelly et al.. The supplementary materials can be safely downloaded by clicking onthis link here. For a graphical summary of the protocol, travel over to the Parts Registry Measurement Section. Note that we WILL NOT be doing Step #1 (synthesis and annealing of oligos to make a test promoter). Instead, we will be testing promoters that have been included with our distribution. COMPLETE PROTOCOL DREW ENDY'S PROTOCOL FOR PRODUCING A STOCK OF BACTERIA Materials 40% glycerol solution Cryogenic vials Method Add 1 ml of 40% glycerol in H2O to a cryogenic vial. Add 1 ml sample from the culture of bacteria to be stored. Gently vortex the cryogenic vial to ensure the culture and glycerol is well-mixed. Alternatively, pipet to mix. Use a tough spot to put the name of the strain or some useful identifier on the top of the vial. On the side of the vial list all relevant information - part, vector, strain, date, researcher, etc. Store in a freezer box in a -80C freezer. Remember to record where the vial is stored for fast retrieval later. Notes While it is possible to make a long term stock from cells in stationary phase, ideally your culture should be in logarithmic growth phase. TK's PREPARING ELECTROCOMPETENT CELLS Electrocompetent cells Protocol from Austin Che and annotated by Reshma Shetty based on online research. Materials Equipment -80°C freezer 37°C incubator Refrigerated centrifuge that accepts 225 mL culture tubes Chemicals and reagents ~500 mL LB Lennox supplemented with appropriate concentration of antibiotic if appropriate. ~600 mL sterile deionized water chilled to 4°C 50 mL sterile 10% glycerol in deionized water chilled to 4°C Ice bucket and ice Dry ice, ethanol bath Supplies Many 1.5 mL plastic tubes chilled to -80 °C 14 mL culture tube for starter culture 2 L flask for culture 225 mL plastic tubes for centrifugation Pipets Procedure Prechill all tubes and pipets at 4°C or -80°C as appropriate. Also rinse all flasks with H2O prior to autoclaving in order to remove residual detergents that may remain on glassware from dishwashing. This step may increase competency. Autoclaving with water, which is then discarded, is even better. Inoculate 5mL LB medium and grow overnight at 37°C with rotation. Use LB Lennox rather than LB Miller in order to lessen salt content of media Add the 5mL overnight culture to 450mL LB medium and incubate at 37°C with vigorous shaking until the OD 600nm is between 0.5 and 1.0. It should take about 3 hours. For recA- strains, the OD 600 nm should be between 0.5 and 0.7 according to one online source. Fast cool the centrifuge with the correct rotor to 4°C Pour the culture into two 225 mL centrifuge tubes. Place the tubes on ice for 15 minutes. This step can vary in incubation time between 15 minutes and 1 hr. Longer incubation times may lead to higher competency. For the following steps it is important to keep cells cold and remove all the supernatant in each step to remove residual ions. Centrifuge for 10 mins at 2000g at 4°C Remove supernatant and gently resuspend pellets with 200mL cold sterile water. Initially add 10-20 mL of water and resuspend by pipetting. Then add the rest of the water. Centrifuge for 15 mins at 2000g at 4°C Remove supernatant and gently resuspend pellets with 200mL cold sterile water. Initially add 10-20 mL of water and resuspend by pipetting. Then add the rest of the water. Hold on ice for 30 minutes Centrifuge for 15 mins at 2000g at 4°C Remove supernatant and gently resuspend pellets with 25mL cold 10% glycerol. This can be optionally transferred to a 50 mL conical tube. Hold on ice for 30 minutes Centrifuge for 15 mins at 1500g at 4°C Remove the supernatant and add 500 μl of 10% glycerol Resuspend the cells in a final volume of approximately 1 ml Aliquot 50 μL per tube (tubes on ice) Shock freeze cell suspensions in a dry ice and ethanol bath. One website recommended against using liquid nitrogen but did not justify this recommendation. Store at -80°C Notes RS 17:49, 13 June 2006 (EDT): Dont' forget to use the conical bottoms on the swinging buckets in the centrifuge ... not the flat ones. Otherwise, your tubes will break. (Oops.) This protocol is for transforming plasmid DNA into Escherichia coli cells. Materials Electrocompetent cells Plasmid DNA (from a ligation reaction) Ice Ice bucket For the following, you need one per DNA sample Electroporation cuvette (either 1mm or 2mm gap width) Electroporator 1.5 mL eppendorf tube LB-agar plate with appropriate antibiotic 1mL SOC at room-temperature Procedure Chill electroporation cuvettes, DNA samples and tubes on ice. Place LB-agar plates in 37°C incubator to warm. Once cuvettes are cold, remove electrocompetent cells from -80°C freezer and thaw on ice. Alternatively, freshly prepared electrocompetent cells may be used immediately. If electrocompetent cells are not already in individual aliquots, then aliquot out into pre-chilled 0.6mL tubes. Turn on electroporator and set voltage to either 1.25 kV (1mm cuvettes) or 2.5 kV (2mm cuvettes). Dial a P2 pipetman to either 1 or 2μL depending on the salt content of your DNA sample and . Use 2μL for samples that have been purified in some way. Dial a P200 pipetman to 50μL or whatever volume of electrocompetent cells you want to use. Usually 20-50μL. Dial a P1000 pipetman to 950μL and pipet in SOC. Place pipetman on counter such that tip doesn't touch anything. Pipet 1-2μL of DNA sample and add to electrocompetent cells. Swirl tip around gently in cells to mix DNA and cells. Do not pipet up and down. Place cells back on ice to ensure they remain cold. Transfer cell-DNA mixture to cuvettes using P200 pipetman. Try not to handle cuvette base too much so that it stays cold. Tap the cuvette on the counter gently so that cells are at the bottom and to remove any air bubbles. Wipe off excess moisture from outside of cuvette. Place in chamber of electroporator. Slide the chamber in so that the cuvette sits snugly between electrodes. Pulse the cells with a shock by pressing button on electroporator. Remove cuvette from the chamber and immediately add SOC. This step should be done as quickly as possible to prevent cells from dying off. Transfer SOC-cell mixture to chilled eppendorf tube. Chill sample on ice for 2 mins to permit the cells to recover. Transfer eppendorf tube to 37°C incubator and shake to promote aeration. Incubate for 1 hr to permit expression of antibiotic resistance gene. Plate transformation onto prewarmed LB-agar plate supplemented with appropriate antibiotic. I generally plate 200μL but appropriate plating volume depends on efficiency of the transformation. Incubate plate overnight at 37°C. Leave remaining SOC-cell mixture on the benchtop overnight. If you don't have any transformants, plate the rest of the transformation in the morning. Notes If you are in a hurry and your selection marker is ampicillin, you can go ahead and plate immediately because ampicillin takes a while to be pumped into cells at a high enough concentration to have an effect. OWW's TOP10 CHEMICALLY COMPETENT CELLS Overview This protocol is a variant of the Hanahan protocol [1] using CCMB80 buffer for DH10B, TOP10 and MachI strains. It builds on Example 2 of the Bloom05 patent as well. This protocol has been tested on TOP10, MachI and BL21(DE3) cells. See Bacterial Transformation for a more general discussion of other techniques. The Jesse '464 patent describes using this buffer for DH5α cells. The Bloom04 patent describes the use of essentially the same protocol for the Invitrogen Mach 1 cells. This is the chemical transformation protocol used by Tom Knight and the Registry of Standard Biological Parts. Materials Detergent-free, sterile glassware and plasticware (see procedure) Table-top OD600nm spectrophotometer SOB CCMB80 buffer 10 mM KOAc pH 7.0 (10 ml of a 1M stock/L) 80 mM CaCl2.2H2O (11.8 g/L) 20 mM MnCl2.4H2O (4.0 g/L) 10 mM MgCl2.6H2O (2.0 g/L) 10% glycerol (100 ml/L) adjust pH DOWN to 6.4 with 0.1N HCl if necessary adjusting pH up will precipitate manganese dioxide from Mn containing solutions. sterile filter and store at 4°C slight dark precipitate appears not to affect its function Procedure Preparing glassware and media Eliminating detergent Detergent is a major inhibitor of competent cell growth and transformation. Glass and plastic must be detergent free for these protocols. The easiest way to do this is to avoid washing glassware, and simply rinse it out. Autoclaving glassware filled 3/4 with DI water is an effective way to remove most detergent residue. Media and buffers should be prepared in detergent free glassware and cultures grown up in detergent free glassware. Prechill plasticware and glassware Prechill 250mL centrifuge tubes and screw cap tubes before use. Preparing seed stocks Streak TOP10 cells on an SOB plate and grow for single colonies at 23°C room temperature works well Pick single colonies into 2 ml of SOB medium and shake overnight at 23°C room temperature works well Add glycerol to 15% Aliquot 1 ml samples to Nunc cryotubes Place tubes into a zip lock bag, immerse bag into a dry ice/ethanol bath for 5 minutes This step may not be necessary Place in -80°C freezer indefinitely. Preparing competent cells Inoculate 250 ml of SOB medium with 1 ml vial of seed stock and grow at 20°C to an OD600nm of 0.3 This takes approximately 16 hours. Controlling the temperature makes this a more reproducible process, but is not essential. Room temperature will work. You can adjust this temperature somewhat to fit your schedule Aim for lower, not higher OD if you can't hit this mark Centrifuge at 3000g at 4°C for 10 minutes in a flat bottom centrifuge bottle. Flat bottom centrifuge tubes make the fragile cells much easier to resuspend It is often easier to resuspend pellets by mixing before adding large amounts of buffer Gently resuspend in 80 ml of ice cold CCMB80 buffer sometimes this is less than completely gentle. It still works. Incubate on ice 20 minutes Centrifuge again at 4°C and resuspend in 10 ml of ice cold CCMB80 buffer. Test OD of a mixture of 200 μl SOC and 50 μl of the resuspended cells. Add chilled CCMB80 to yield a final OD of 1.0-1.5 in this test. Incubate on ice for 20 minutes Aliquot to chilled screw top 2 ml vials or 50 μl into chilled microtiter plates Store at -80°C indefinitely. Flash freezing does not appear to be necessary Test competence (see below) Thawing and refreezing partially used cell aliquots dramatically reduces transformation efficiency by about 3x the first time, and about 6x total after several freeze/thaw cycles. Measurement of competence Transform 50 μl of cells with 1 μl of standard pUC19 plasmid (Invitrogen) This is at 10 pg/μl or 10-5 μg/μl This can be made by diluting 1 μl of NEB pUC19 plasmid (1 μg/μl, NEB part number N3401S) into 100 ml of TE Hold on ice 0.5 hours Heat shock 60 sec at 42C Add 250 μl SOC Incubate at 37 C for 1 hour in 2 ml centrifuge tubes rotated using 2ml centrifuge tubes for transformation and regrowth works well because the small volumes flow well when rotated, increasing aeration. For our plasmids (pSB1AC3, pSB1AT3) which are chloramphenicol and tetracycline resistant, we find growing for 2 hours yields many more colonies Ampicillin and kanamycin appear to do fine with 1 hour growth Plate 20 μl on AMP plates using sterile 3.5 mm glass beads Good cells should yield around 100 - 400 colonies Transformation efficiency is (dilution factor=15) x colony count x 105/µgDNA We expect that the transformation efficiency should be between 5x108 and 5x109 cfu/µgDNA 5x Ligation Adjustment Buffer Intended to be mixed with ligation reactions to adjust buffer composition to be near the CCMB80 buffer KOAc 40 mM (40 ml/liter of 1 M KOAc solution, pH 7.0) CaCl2 400 mM (200 ml/l of a 2 M solution) MnCl2 100 mM (100 ml/l of a 1 M solution) Glycerol 46.8% (468 ml/liter) pH adjustment with 2.3% of a 10% acetic acid solution (12.8ml/liter) Previous protocol indicated amount of acetic acid added should be 23 ml/liter but that amount was found to be 2X too much per tests on 1.23.07 --Meaganl 15:50, 25 January 2007 (EST) water to 1 liter autoclave or sterile filter Test pH adjustment by mixing 4 parts ligation buffer + 1 part 5x ligation adjustment buffer and checking pH to be 6.3 - 6.5 Reshma 10:49, 11 February 2008 (CST): Use of the ligation adjustment buffer is optional. References Hanahan D, Jessee J, and Bloom FR. Plasmid transformation of Escherichia coli and other bacteria. Methods Enzymol. 1991;204:63-113. DOI:10.1016/0076-6879(91)04006-a | Reusch RN, Hiske TW, and Sadoff HL. Poly-beta-hydroxybutyrate membrane structure and its relationship to genetic transformability in Escherichia coli. J Bacteriol. 1986 Nov;168(2):553-62. DOI:10.1128/jb.168.2.553-562.1986 | Addison CJ, Chu SH, and Reusch RN. Polyhydroxybutyrate-enhanced transformation of log-phase Escherichia coli. Biotechniques. 2004 Sep;37(3):376-8, 380, 382. DOI:10.2144/04373ST01 | US Patent 6,709,852 Media:pat6709852.pdf US Patent 6,855,494 Media:pat6855494.pdf US Patent 6,960,464 Media:pat6960464.pdf MINIPREP (BASED ON THE QIAGEN KIT) Materials For purifying plasmid DNA from Escherichia coli cells, the Qiagen Spin Miniprep Kit produces quite reliable results. Do not autoclave solutions containing isopropanol or MOPS; use sterile filtration if necessary. Buffer P1 50 mM Tris-HCl pH 8.0 10 mM EDTA 100 μg/ml RNaseA The buffer and RNaseA can also be ordered from Qiagen separately (catalog numbers 19051 and 19101). Buffer P2 200 mM NaOH 1% SDS Buffer P3 (not for spin columns, but for Qiatips, midi, maxi, giga kits) 3.0 M potassium acetate pH 5.5 Buffer N3 4.2 M Gu-HCl 0.9 M potassium acetate pH 4.8 Buffer PB 5 M Gu-HCl 30% ethanol (maybe add 10mM Tris-HCL PH 6.6, and that is better) Buffer PE 10 mM Tris-HCl pH 7.5 80% ethanol Buffer QBT equilibration buffer 750 mM NaCl 50 mM MOPS pH 7.0 15% isopropanol 0.15% triton X-100 Buffer QC wash buffer 1.0M NaCl 50 mM MOPS pH 7.0 15% isopropanol Buffer QF elution buffer 1.25M NaCl 50 mM Tris-HCl pH 8.5 15% isopropanol Buffer QN 1.6M NaCl 50 mM MOPS pH 7.0 15% isopropanol Buffer FWB2 1M potassium acetate, pH 5.0 (Source: [1], US Patent 6,383,393) The LyseBlue indicator dye added to some of the buffers is Thymophthalein, pH shift from colorless to blue at pH 9.3 Protocol See here or here for the handbook for the Qiagen Spin Miniprep Kit. If you have never done this protocol before, read the the background information in the handbook (like the Important Notes section). It contains useful information. The following has been reproduced from the handbook and annotated based on experience with the kit. Protocol: QIAprep Spin Miniprep Kit Using a Microcentrifuge This protocol is designed for purification of up to 20 μg of high-copy plasmid DNA from 1–5 ml overnight cultures of E. coli in LB (Luria-Bertani) medium. For purification of low-copy plasmids and cosmids, large plasmids (>10 kb), and DNA prepared using other methods, refer to the recommendations on page 37. Please read “Important Notes” on pages 19–21 before starting. Note: All protocol steps should be carried out at room temperature. Procedure Resuspend pelleted bacterial cells in 250 µl Buffer P1 (kept at 4 °C) and transfer to a microcentrifuge tube. Ensure that RNase A has been added to Buffer P1. No cell clumps should be visible after resuspension of the pellet. Add 250 μl Buffer P2 and gently invert the tube 4–6 times to mix. Mix gently by inverting the tube. Do not vortex, as this will result in shearing of genomic DNA. If necessary, continue inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for more than 5 min. Add 350 μl Buffer N3 and invert the tube immediately but gently 4–6 times. To avoid localized precipitation, mix the solution gently but thoroughly, immediately after addition of Buffer N3. The solution should become cloudy. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge. A compact white pellet will form. Apply the supernatants from step 4 to the QIAprep spin column by decanting or pipetting. Centrifuge for 30–60 s. Discard the flow-through. Spinning for 60 seconds produces good results. (Optional): Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30–60 s. Discard the flow-through. This step is necessary to remove trace nuclease activity when using endA+ strains such as the JM series, HB101 and its derivatives, or any wild-type strain, which have high levels of nuclease activity or high carbohydrate content. Host strains such as XL-1 Blue and DH5α™ do not require this additional wash step. Although they call this step optional, it does not really hurt your yield and you may think you are working with an endA- strain when in reality you are not. Again for this step, spinning for 60 seconds produces good results. Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30–60 s. Spinning for 60 seconds produces good results. Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer. IMPORTANT: Residual wash buffer will not be completely removed unless the flow-through is discarded before this additional centrifugation. Residual ethanol from Buffer PE may inhibit subsequent enzymatic reactions. They are right about this. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min. If you are concerned about the concentration of the DNA, you can alternatively add 30 μL water to the center of the column, incubate at room temperature on the bench for 5 mins and then centrifuge for 1 min. This will increase the concentration of DNA in your final sample which can be useful in some cases. See notes below for why you should elute in water rather than the Buffer EB they recommend if you plan to sequence your sample. Even if you are not sequencing, it may be beneficial to elute in water. For instance, if you elute in buffer EB and you are using this DNA in a restriction digest, then the additional salts in your sample can affect the salt content of your digest. This may matter with some finicky enzymes. Notes If you are doing more than ~10 minipreps simultaneously, it can save time to switch to the vacuum manifold version of this protocol since you eliminate having to load and unload samples into the centrifuge. The sequencing center has begun using new machines and as a result you may want to consider eluting in water rather than EB. See note from sequencing center. The elution is dependent on pH, however measuring the pH of unbuffered water is difficult. However, anecdotally we have been able to get good yields using the water from the stock room. Eluting in deionized water from the Knight lab has also produced good results. I use the "mini-fuge" for the binding and washing steps. You still have to do the drying step after the PE wash in a "real" microfuge though. Passing the lysate over the column twice increases yield by about 20%. Contaminating salt from the initial lysate or the PB will ruin a sequencing reaction more frequently than eluting in the EB (10 mM Tris as a small component of the total sequencing reaction is negligible). I always elute with EB and my reactions sequence just dandy. There are two major sources of salt contamination: the inside upper edge of the spin column and the residual PB mixing with the PE wash. When you add the initial PE, it mixes with the leftover junk in the column. Spinning this through can only lower the salt to a level that was present after mixing. To get around these problems, I do two PE washes of about 300-500 μL. For the the first, I dispense the liquid from the pipette tip along the inner ledge of the spin column in a circular motion to wash off the residue there. I follow the first PE wash with a second to further de-salt the sample before the drying spin. Yes, it adds a step, but the time spend here is far less than waiting three days only to find out your sequencing didn't work. Heating the elution buffer to 55°C prior to loading on the column can slightly increase yields. Similarly, doing the elution in two steps (first a 30 μL elution and then a 20 μL dilution) can also slightly increase yields. BioCoder version Following is the Miniprep/Qiagen kit protocol in BioCoder, a high-level programming language for expressing biology protocols. What you see here is the auto-generated text ouput of the protocol that was coded up in BioCoder (see Source code). More information about BioCoder can be found on my home page. Feel free to mail me your comments/ suggestions.Vaishnavi Text Output Miniprep/Qiagen kit protocol Source Code Miniprep/Qiagen kit protocol - source code QIAGEN BUFFERS AND COLUMN RECYCLING Clicking the link above will provide you with the compositions of the different buffers used by Qiagen, as well as instructions on how to conserve the column that are included with the kit. NEB/GINKGO'S 3A BIOBRICK ASSEMBLY MANUAL The above link brings you to the protocol we will use for the processes of 3A standard assembly (restriction digest and ligation of Biobrick plasmids). TK's RESTRICTION DIGEST Materials Restriction enzymes (EcoR I, Spe I, Xba I or Pst I) from NEB NEB2 buffer BSA Deionized, sterile H2O Digest Mix Example - 50 μL reaction. 100 μL reactions are also common especially if your DNA to be cut is dilute. 5 μL NEB2 buffer (for all digests with BioBricks enzymes, we use NEB2 buffer. It keeps things simple and seems to work). X μL DNA (usually ~500 ng depending on downstream uses). 0.5 μL 100X BSA (added to all digests because BSA never hurts a restriction digest) 1 μL BioBricks enzyme 1 (regardless of the volume of the reaction, 1 μL enzyme is used because generally this represents a 10-25 fold excess of enzyme and is therefore sufficient for most digests. Also, it can be difficult to accurately pipet less than 1 μL of enzyme since it is sticky due to the glycerol content.) 1 μL BioBricks enzyme 2 (42.5 - X) μL deionized, sterile H2O Procedure 1. Add appropriate amount of deionized H2O to sterile 0.6 mL tube 2. Add restriction enzyme buffer to the tube. Vortex buffer before pipetting to ensure that it is well-mixed. 3. Add BSA to the tube. Vortex BSA before pipetting to ensure that it is well-mixed. 4. Add appropriate amount of DNA to be cut to the tube. Vortex DNA before pipetting to ensure that it is well-mixed. 5. Add 1 μL of each enzyme. Vortex enzyme before pipetting to ensure that it is well-mixed. Also, the enzyme is in some percentage of glycerol which tends to stick to the sides of your tip. To ensure you add only 1 μL, just touch your tip to the surface of the liquid when pipetting. 6. Place in thermal cycler (MJ Research, PT-200) and run digest protocol. 4-6 hour incubation at 37°C Use a longer incubation time if you have time or are worried about the efficiency of cutting. I think this time can be shortened to 2 hrs while still cutting to completion. 20 mins at 80°C to heat inactivate enzyme. This step is sufficient to inactivate even Pst I. 4°C forever (or until you pull the reaction out of the thermal cycler). 7. Generally, use some method of DNA purification to eliminate enzymes and salt from the reaction.