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| =How Do BioBricks Work?= | | =Synthetic Biology= |
| A Biobrick is a piece of DNA flanked by a STANDARD set of restriction sites: EcoRI (E), NotI (N), XbaI (X) upstream to the left; SpeI (S), NotI (N), PstI (P) downstream to the right.
| | *[[IGEM:Harvard/2010/General Protocols/BioBricks | How Do BioBricks Work?]] |
| | *[[Silver: BB Strategy | Strategy for Making Parts]] |
| | *[[Silver: PCR | PCR]] |
| | *[[Silver: Oligonucleotide Inserts | Oligonucleotide Inserts]] |
| | *[[Silver: Site-Directed Mutagenesis | Site-Directed Mutagenesis]] |
| | *[[Silver: Restriction Digest | Restriction Digest]] |
| | *[[IGEM:Harvard/2010/General Protocols/DNApurification | DNA Gel Electrophoresis and Fragment Purification]] |
| | *[[IGEM:Harvard/2010/General Protocols/Nanodrop | Measuring DNA Concentration Using a Nanodrop Spectrophotometer]] |
| | *[[Silver: Ligation | Ligation]] |
| | *[[Silver: Quick & Dirty Digest & Ligation | Quick & Dirty Digest & Ligation]] |
| | *[[Silver: Bacterial Transformation | Bacterial Transformation]] |
| | *[[Silver: Plasmid Verification | Plasmid Verification]] |
| | *[http://labs.fhcrc.org/fero/Protocols/RNeasy_Mini_Handbook.pdf Qiagen RNeasy Plant Mini Kit] |
| | *[http://cshprotocols.cshlp.org/cgi/reprint/2007/4/pdb.kit23.pdf iScript cDNA Synthesis] |
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| *Standard Assembly of BioBricks is explained on the [http://partsregistry.org/Assembly:Standard_assembly Parts Registry Website] | | =Microbiology= |
| | *[[IGEM:Harvard/2010/General Protocols/EColi | Growing E. coli]] |
| | *[[IGEM:Harvard/2010/General Protocols/Agrobacterium | Growing Agrobacterium]] |
| | *[[IGEM:Harvard/2010/General Protocols/CompetentAgrobacterium | Making Electro-Competent Agrobacterium]] |
| | *[[IGEM:Harvard/2010/General Protocols/Agrobacterium Electroporation | Electroporating Agrobacterium]] |
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| *The sequence of the BioBrick cloning site is: gaattc(E) gcggccgc(N) tctag(X) [BioBrick Part] actagt(S) gcggccg(N) ctgcag(P)
| | =Plants= |
| | | *[[IGEM:Harvard/2010/General Protocols/Arabidopsis | Growing Arabidopsis]] |
| *E-N-X are referred to as the "BioBrick Prefix" and S-N-P are referred to as the "Biobrick Suffix"
| | *[[IGEM:Harvard/2010/General Protocols/FlowerDipping | Arabidopsis transformation via Flower Dipping]] |
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| *The BioBrick part itself must NOT contain any of the standard restriction sites, or else it will be chopped up into pieces during assembly
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| *The Biobrick vector we will be using is called V0120, a high-copy plasmid (~300 ng/uL from a mini-prep of a 5 mL culture)
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| =Growing a Bacterial E. coli Culture= | |
| E. coli is a typical bacteria used for molecular cloning. Long-term storage of BioBrick plasmids is in E. coli suspended in glycerol (500ul cells:500ul glycerol) and kept at -80˚C. Growing E. coli is performed at 37˚C, at which temperature they divide every 20 minutes.
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| *Day1: Making streaks from glycerol stocks | |
| **Warm agar plate at 37˚C for 10 minutes
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| **Label lid of plate with what you're streaking on it, and your name, and the date
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| **Locate desired glycerol stock in -80˚C freezer and, using a sterile toothpick, scrape out a tiny bit and streak on plate
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| **Incubate plate overnite at 37˚C
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| *Day2: Growing liquid cultures
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| **Label 15mL falcon tube and add 5mL of LB media + selective antibiotic
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| **Using 200uL tip w/o filter, touch streak or single colony and put tip in falcon tube
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| **Grow culture overnite in a shaking 37˚C incubator
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| =Plasmid Minipreps: Qiagen Miniprep Kit=
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| To extract plasmid DNA from bacteria, perform a miniprep using the Qiagen miniprep protocol.
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| =DNA Digests=
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| Use appropriate enzyme to cut the BioBrick plasmids or parts to be ligated, taking care as to whether you're performing a "front-end" or "back-end" ligation.
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| *Write out exactly what you're building, i.e. part names, sizes, etc
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| *In a 1.5mL centrifuge tube, set up digest reaction as follows:
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| **Plasmid DNA: ____ uL = ~1000ng
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| **Enzyme 1: 1.0 uL
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| **Enzyme 2: 1.0 uL
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| **Buffer: 2.0 uL
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| **Water: ____ uL
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| ____________________________________
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| **Total: 20.0 uL
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| (Note: Always be sure that the TOTAL enzyme concentration is never >10% of TOTAL volume, i.e. 2uL enzymes/20uL total reaction is 10%.)
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| *Mix tube well and digest at 37˚C for at least 5 minutes. If you are including a phosphotase enzyme, you need to deactivate it by next incubating the tube at 65˚C for 10 minutes.
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| *Proceed to gel purification.
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| =DNA Gel Electrophoresis and Fragment Purification=
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| *****NOTE: Ethidium Bromide (EtBr) is a mutagen ---- always wear gloves when handling it*****
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| *Making an agarose gel
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| **To make a 1% gel, add 1g agarose to 100mL 1x TAE buffer in 200mL flask
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| **Microwave for 1.5 minutes
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| **Swirl and check that the agarose is completely dissolved and there are no air bubbles
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| **Allow gel solution to cool for a bit
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| **While gel is cooling, tape the ends of gel mold so gel liquid cannot escape. Don't forget the combs!! Sit mold on a flat surface.
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| **Add 4.5ul of EtBr to 100mL of gel and mix by swirling (avoid making bubbles)
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| **Pour into taped gel mold and allow to cool until solid
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| =Measuring DNA Concentration using a Nanodrop Spectrophotometer=
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| *Open Nanodrop software and choose "nucleic acids"
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| *Wipe Nanodrop pedestal with distilled water to clean it
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| *Pipette 2uL of water onto the pedestal and lower the arm gently
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| *Initialize the machine and wait for it to finish (you'll hear a couple of clicks)
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| *Raise arm and wipe water off of pedestal with Kimwipe
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| *Pipette 2uL of appropriate buffer (whatever you eluted you DNA into at the end of the DNA miniprep - typically EB Buffer) onto pedestal and lower arm
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| *Click "Blank" and wait for machine to finish measuring
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| *Repeat last 3 steps using your DNA samples and clicking "Measure" instead of "Blank." Record the absorbance (A260), purity (A260/A280), and concentration (ng/uL) for each sample
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| =Ligation Reaction=
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| =Transformation of E. coli=
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| =Confirming the Assembly=
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| =Growing Arabidopsis=
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| =Growing Agrobacterium=
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| We have received from Amy Hark the agrobacterium strain: GV3101 pMP90. This is the strain that was used to transform the plant vectors we purchased into Arabidopsis.
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| *Frequently used for many binary vectors for Arabidopsis | |
| *Resistance to Rif (10mg/L) (in genome), Gent (30mg/L) (in helper plasmid), and possibly Kan (30mg/L) (in helper plasmid) ---- she told me she usually only selects with Gent, but we may want to try and see if it is still resistant to Kan
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| *Known to develop spontaneous Tet resistance, so this strain should be used with care.
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| *Fast growing colonies after transformation are false. True transformants appear 1-2 days after fast growing colonies.
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| *Use 100ug/mL timentin/cefotaxim for Agro removal when selecting transgenic plants on plate
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| *Use bromin to kill agro.
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| =Making Agrobacterium Electro-Competent Cells=
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| This protocol should be doubled up - it is almost the same amount of work and you can get some 80 tubes.
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| *Inoculate colony O/N in 3mL LB+antibiotics at 30˚C shaker
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| *Transfer O/N cultures to 200mL LB in sterile 500mL flask and shake at 250rpm until the OD is 0.3 (4-5h)
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| *Spin in sterile screw cap tubes at 4˚C, 5000 rpm for 10min. Check to make sure cells are pelleted - if not repeat at higher speed
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| *Aspirate supernatant, resuspend pellet in 20mL ice cold 1mM HEPES pH7 (sterile filtered), re-spin. Repeat this step 2 more times.
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| *After aspirating, resuspend pellet in 2mL ice cold 10% glycerol (sterile filtered)
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| *ASAP dispense in 40ul aliquots in pre-chilled, sterile eppis, freeze in liquid nitrogen and store at -70˚C
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| =Arabidopsis transformation via Flower Dipping=
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| *Grow healthy Arabidopsis plants until they are flowering. Grow under log days in pots in soil covered with bridal veil, window screen or cheesecloth.
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| *(Optional) Clip first botls to encourage proliferation of many secondary bolts. Plants will be ready 406 days after clipping. Clipping can be repeated to delay plants. Optimal plants have many immature flower clusters.
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| *Grow 3mL O/N cultures of construct in AgroGV3101 with proper antibiotics selection
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| *Inoculate into 250mL LB kan/gent (does 4-6 pots)
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| *Spin down, 7k X 5min
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| *Resuspendto OD600=0.8 (can be higher or lower) in 5% sucrose, 0.05% Silwet L-77 (50g sucrose, 0.5mL Silwet, 1L distilled water). If there are problems with toxicity, use 0.02% or as low as 0.005%. You will need 100-200mL for each two or three small pots to be dipped, or 400-500 mL for each 2-3 3.5" (9cm) pots.
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| *Dip above ground parts of plant in solution for 2-3 seconds, with gentle agitation. You should then see a film of liquid coating plant.
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| *Place dipped plants under a dome or cover for 16-24h to maintain high humidity (plants can be laid on their side if necessary). Do not expose to excessive sunlight.
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| *Water and grow plants normally, tying up loose bolts by some means. Stop watering as seeds become mature.
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| *Harvest dry seed. Tranformatns are usually all independent, but are guarantted to be independent if they come off of separate plants.
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| *Select for transformants using antibiotic or herbicide selectable marker on agar plates. For example, vapor-phase sterilize and plate 40mg = 2000 seed (resuspended in 4mL 0.1% agarose) on .5X MS/0.8% tissue culture Agar plates with antibiotic, cold treat for 2 days, and grow under continuous light for 7-10 days.
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| *Transplant putative transformants to soil. Grow, test and use.
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| For higher rates of transformation, plants may be dipped 2-3 times at 7 day intervals. It is suggested to do one dip 2 days after clipping, and a second dip one week later. Do not dip less than 6 days apart.
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