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Assignments Due

• Gallivan (Curr Opin Chem Biol 2007, 11:612-619)
• Elowitz (Nat Rev Microbiol 2009, 7(5):383-392)
• Introduction to BioBrick™ Standard Biological Parts: read ALL parts under An Introduction to BioBrick™ Standard Biological Parts EXCEPT for "A Team Experience Tutorial"; read "Standard Assembly" under BioBrick™ Assembly; read "How to perform a construction" under At the Bench
• Homework Assignment: The Registry

Lecture Topics

• (Invited Speaker)
• BioBrick™ Standard Assembly

Overview of Experiment

BioBricks are joined using standard cloning techniques and a process called BioBrick Standard Assembly. After treating the digested vector with phosphatase, both the vector and insert DNA will be gel purified.  The insert will be ligated to the vector, and electrocompetent cells will be transformed with the ligation reaction.

Alkaline phosphatase treatment of vector DNA

1. Thaw BBa_R0040 digest at 37°C ("dry" heat block)
2. Add 1/10 volume (3 µl) of 10X antarctic phosphatase reaction buffer
3. Add 1 µl antarctic phosphatase (NEB, catalog # M0289S) to BBa_R0040 digest ONLY and mix
4. Incubate 60 minutes at 37°C ("dry" heat block)
5. Heat inactivate 20 minutes at 80°C
6. Proceed to agarose gel purification of vector and insert

Gel purification of vector and insert DNA

Agarose gel electrophoresis

1. Prepare a wide mini 1% agarose gel with a 15-well comb (use 150 ml) as on Day 1 [pour one gel per 4-6 people]
2. Pulse spin the digest reactions/uncut controls and add 6 µl 6X LB to each
3. Slowly load ALL of each reaction on the gel
4. Load 10 µl NEB Quick-Load 1 kb DNA Ladder
5. Run the gel at 130 V for 30 minutes
6. Photograph the gel and compare the observed bands to the standards
   
   Expected products:
   
   
   BBa_R0040 (promoter/tetR repressible, in pSB1A2, AmpR)
   • Uncut control = supercoiled (cannot estimate size)
   • SpeI/PstI digest = < 2133 bp (small spacer between enzyme sites is removed)
   
   
   BBa_E0840 (green fluorescent protein (GFP) generator, in pSB1A2, AmpR)
   • Uncut control = supercoiled (cannot estimate size)
   • XbaI/PstI digest = 2079 bp, 878 bp

Gel purification (Zymoclean Gel DNA Recovery Kit™)

1. Locate the insert (878 bp band from E0840) and vector (band from R0040) DNA bands on the agarose gel using long wave ( >300 nm) UV light
   
   Wear safety glasses or a face shield to protect your eyes and minimize exposure time to skin
   
   
2. Excise the DNA from the gel using a razor blade or scalpel (use a fresh one for each piece of DNA; dispose of in a sharps container)
   
   cut as small a piece of agarose as possible -- trim off excise gel around the band
   
   
3. Transfer each gel piece to a sterile 1.5 ml tube
4. Pulse spin for ~ 10 sec to "pellet" the agarose (for estimation of gel volume)
5. Add 3 volumes of ADB Buffer™ to each volume of excised agarose
6. Incubate at 55°C until the agarose is completely dissolved (5-10 minutes)
7. Add the melted agarose solution to a spin column in a collection tube
8. Centrifuge at 16,000 x g for 30 seconds and discard column flow-through
9. Add 200 µl wash buffer (contains ethanol) and centrifuge as in step 8 (do not discard flow-through)
10. Repeat the wash and centrifuge as in step 8; put column in a sterile 1.5 ml tube
11. Add nuclease-free water to center of column
    • 15 µl for vector
    • 12 µl for insert
12. After 1 minute, centrifuge at 16, 000 x g for 30-60 seconds to elute DNA
    ***The gel-purified DNA can be used directly in ligations, digests, PCR, etc.***
    
13. Proceed to the ligation

Ligation

T4 DNA ligase is an enzyme encoded by the T4 bacteriophage that "ligates" DNA molecules by covalently joining a 3'-OH to an adjacent 5'-phosphate group. The joined ends may be from a single DNA molecule or from different molecules. Molecules with protruding single strand ends can be ligated together if the ends are compatible (i.e. complementary), so that they can anneal to each other. It is also possible to ligate any two blunt-ended DNA molecules together, although this is considerably less efficient since there is nothing to hold the DNA molecules next to each other. Ligations are used in our experiment to create stable recombinant DNA molecules for use in transformations.

Ligations require planning. Restriction fragments with protruding ends to be ligated must contain compatible complementary sequences. If orientation of an insert is important, two different ends will increase the probability of the correct orientation. The joining of a blunt end to a sticky end can be achieved by converting the sticky end to a blunt end, either by filling in the missing bases of a 5' protruding end using the Klenow fragment or by chewing off a 3' overhang with T4 DNA polymerase (the polymerase has a 3'-5' exonuclease activity used to correct misincorporation of nucleotides).

The components to be ligated are mixed in a ratio determined by the desired product. If recircularization (intramolecular ligation) is the goal, the concentration of fragments is kept low to decrease the probability of two different molecules contacting each other. If a product is to be inserted, such as in a cloning procedure, an excess of insert of 2 to 3 x the vector concentration is used, and the concentration of DNA is higher to increase the occurrences of intermolecular ligation.

Ligation Procedure for LigaFast™ Rapid DNA Ligation System (Promega)

A 1:2 or 1:3 molar ratio of vector:insert DNA is generally recommended when subcloning a DNA fragment into a plasmid vector
Typical ligation reactions use 25-100 ng of vector DNA
Molar ratios are converted to mass ratios using this formula:
[(ng vector x kb size of insert) / kb size of vector] x (molar ratio of insert / vector) = ng of insert
NOTE: the concentration of DNA recovered from an agarose gel is usually quite dilute; consequently, you will use the maximum volume of total DNA (4 µl) for the ligation reaction

Vector: linearized BBa_R0040, ~2133 bp
Insert: 878 bp fragment from XbaI/PstI digest of BBa_E0840


1. Thaw Rapid Ligation Buffer, 2X RLB (60 mM Tris-HCl, pH 7.8; 20 mM MgCl2; 20 mM DTT; 2 mM ATP; 10% PEG) at room temperature; the ligase buffer contains ATP and Mg2+ necessary for the reaction
2. Combine 1 µl vector DNA with 3 µl insert DNA in a 1.5 ml tube
3. Add 5 µl of Rapid Ligation Buffer (2x RLB) followed by 1 µl of T4 DNA ligase (lig) (3u/µl)
4. Mix and pulse spin
5. Incubate the reactions at room temperature for 5 min (for cohesive-ends)

DNA Clean-Up

1. Add 100 µl DNA binding buffer to each ligation reaction
2. Mix by vortexing
3. Transfer to a Zymo-Spin™ column in a collection tube
4. Centrifuge at 16,000 x g for 30 seconds and discard column flow-through
5. Add 200 µl wash buffer to the column
6. Centrifuge at 16,000 x g for 30 seconds
7. REPEAT steps 5 & 6
8. Add 200 µl 80% ethanol and centrifuge as above
   ***This additional wash removes any remaining salts***
9. Transfer the column to a sterile 1.5 ml tube and add 25 µl nuclease-free water directly to the column matrix
10. Wait one minute and centrifuge at 16,000 x g for 30 seconds to elute the DNA

Transformation

1. Prechill 0.1 cm electroporation cuvettes (x3) on ice
2. Thaw the electroporation-competent cells (TetS or TetR) on ice
   
   You must be extremely gentle when working with competent cells. These cells are highly sensitive to temperature changes and/or mechanical lysis. Mix cells by gently tapping the tube or swirling with a pipet tip, not by pipetting up & down or vortexing.
   
   
3. Add ALL of each cleaned-up ligation reaction to a tube of cells and gently mix; incubate on ice for 1 minute
   
4. Place the cuvette on its side and add 1) cells+R/E ligation; 2) cells+R ligation; or 3) cells ONLY (NO DNA added)  [save the tubes! put on a rack at room temp.]
5. Gently tap the cuvette until the mixture of cells and DNA settles evenly to the bottom (i.e., there is no gap across the cuvette)
6. Wipe outside of cuvette with KimWipe and slide the cuvette into the electroporation chamber until the cuvette connects with the electrical contacts
7. Pulse sample ONCE at 1.8 kV, 200 ohms, 25 µF (Bio-Rad Electroporator)
   **Record the time constant (in ms) and the actual volts (kV) delivered to sample**
   
   time constant (τ): the amount of time required for the actual voltage of the delivered pulse to decay to 1/e (37%) of the initial voltage
   {τ = Resistance (R) x Capacitance (C)}
   
   
8. QUICKLY remove the cuvette from the chamber and add 1 ml of prewarmed (40-44°C) sterile SOC medium to the cells
9. With a sterile Pasteur pipet, quickly but gently resuspend the cells and transfer the cell suspension to the original 2 ml tube
10. Incubate the sample at 37°C for 1 hour with shaking at 225 rpm
11. Pipet 175 µl of sterile LB on a LB-ampicillin plate and add 25 µl of transformed cells to the pool of LB
12. Pour 10 - 20 sterile solid glass beads onto the plate, set the plate on the benchtop, and "shake" plate in a perpendicular motion; invert plate to pour off beads (collect in a large beaker -- these can be reused)
13. On a second LB-amp plate, pipet 100 µl of sterile LB and add 100 µl of transformed cells
14. REPEAT step 12
15. On a third LB-amp plate, add 100 µl of control ligation transformation (i.e., cells+R ligation)
16. REPEAT step 12
17. On a fourth LB-amp plate, add 100 µl of negative control transformation (i.e., NO DNA)
18. REPEAT step 12
19. Incubate the plates (upside down) overnight at 37°C
    
    

Copyright, Acknowledgements, and Intended Use
Created by B. Beason (bbeason@rice.edu), Rice University, 21 November 2007
Updated 4 November 2009