20.109(S11):Ligate DNA and transform bacteria (Day4)

Introduction
Today you will ligate your linearized and purified plasmid backbone with your unique annealed oligos. The reaction requires ATP and the enzyme T4 DNA ligase. During the ligation, hydrogen bonds will form between the overhangs on the fragments, and then the ligase will repair the phosphate backbone, creating a stable circular plasmid (as shown in the figure below). A 5' phosphate is required for the ligation to work well; thus, the oligos we ordered from IDT were prepared with this modification.

If all goes well, your ligation reactions will generate your desired construct: the pED-IPTG-INS backbone carrying a modified Plux-&lambda; insert. However, the DNA is present at a low concentration, and we would like to amplify it. Thankfully, we have E. coli bacteria to do this for us quite efficiently! Bacteria can take up foreign DNA in a process called transformation, during which a single plasmid enters a bacterium and, once inside, replicates and expresses the genes it encodes. Most bacteria do not exist in a transformation-ready state, but can be made permeable to foreign DNA by chemical treatment or other means. Cells that are capable of transformation are referred to as competent. Competent cells are extremely fragile and should be handled gently, i.e., kept cold and not vortexed. Bacterial transformation is efficient enough for most lab purposes, resulting in as many as 109 transformed cells per microgram of DNA, but even with highly competent cells only 1 DNA molecule in about 10,000 is successfully transformed. Thus we need a way to identify transformed cells, which is usually accomplished with antiobiotics. For example, the parent vector for the pED plasmids, psB4A3, also carries a gene that leads to ampicillin-resistance. Consequently, a transformed bacterium will grow on ampicillin-containing agar medium, while untransformed cells will die before they can form a colony (see figure above right).







Protocols
Before you start today, you might take a moment to make a plan for the afternoon with your partner. Besides the options listed in Part 1, Step 2 below, one other approach may work well. First, prepare for your &beta;-gal assay but don't do lyse the cells or add ONPG yet. Then, set up your ligation. That way, you should definitely be able to finish the &beta;-gal assay during the 1 hour that you have.

Part 1: Prepare ligation reactions
You will prepare two ligation reactions, one with both backbone and insert, and one with only backbone. The contents will be:

^New England Biolabs sells 10X Ligation buffer to use with their ligase. It contains ATP so must be kept on ice.

If you cannot meet all of the requirements above given your backbone concentration, it may be okay to use less than 50 ng of it, or to change the ratio of backbone to insert. Please see the teaching faculty to discuss how to proceed.


 * 1) Assemble the reactions in eppendorf tubes but not in the order listed. Please ask if you are unsure in what order to assemble the components.
 * 2) When the ligation mixes are complete, flick the tubes to mix the contents, quick spin them in the microfuge to bring down any droplets, then incubate the reactions at room temperature for at least 1 hour.
 * 3) *Even if you will only incubate 1 hour, begin preparing Part 2 (e.g., measure OD600 values and aliquot Z-buffer into tubes). You can finish Part 2 during a later incubation or after you have finished the transformation.
 * 4) *Alternatively, you can continue the ligation reaction for more than 1 hour until you have finished Part 2. Just keep in mind that there is a later half-hour incubation in Part 3 as well.
 * 5) *Finally, you and your partner can split up some of the work if that helps, especially since you have all done &beta;-gal assays before.
 * 6) Place the reactions on the 65 &deg;C heat block for 10 minutes to inactivate the enzyme.

Part 2: Transfer function for IPTG &rarr; lacZ

 * 1) Follow the protocol from Day 2 for performing &beta;-gal assays. When your reactions are stopped and spun down, transfer to a 96-well plate (previously described as Option 2).
 * 2) *Remember to order your samples from least to most expected activity, especially if one person is working without a partner during the ONPG addition step.

Part 3: Transform bacteria with ligated DNA
You will perform 4 bacterial transformations: one for each of the ligation mixtures, one mock transformation, and one transformation with 5 ng of intact plasmid DNA to assess transformation frequency.


 * 1) Prewarm and dry 4 LB+AMP plates by placing them in the 37&deg;C incubator, media side up with the lids ajar.
 * 2) Get an aliquot of competent cells from one of the teaching faculty. Keep these cells on ice at all times. There should be at least 200 &mu;l of cells in each tube. Aliquot 50 &mu;l of cells into 4 clean eppendorf tubes.
 * 3) Add DNA to each tube of cells as indicated below.
 * 4) *Ligation reactions, 5 &mu;L of appropriate mixture to each
 * 5) *Positive control, 1 &mu;L of pED-IPTG-INS
 * 6) *Negative control, none
 * 7) Flick to mix the contents and leave the tubes on ice for at least 5 minutes.
 * 8) Heat shock the cells at 42&deg;C for 90 seconds exactly and put on ice for two minutes. Use your timer.
 * 9) Add 0.5 mL of warm LB to each sample, then move them to the 37 &deg;C incubator.
 * 10) Allow the cells to recover and begin expressing ampicillin resistance for 30 minutes. At the same time, pre-warm and dry four LB+AMP plates by placing them in the 37&deg;C incubator, media side up with the lids ajar.
 * 11) Plate 250 &mu;L of each transformation mix on LB+AMP plates. After dipping the glass spreader in the ethanol jar, you should pass it through the flame of the alcohol burner just long enough to ignite the ethanol. After letting the ethanol burn off, the spreader may still be very hot, and it is advisable to tap it gently on a portion of the agar plate without cells in order to equilibrate it with the agar (if it sizzles, it's way too hot). Once the plates are ready, wrap them together with one piece of colored tape and incubate them in the 37&deg;C incubator overnight. One of the teaching faculty will remove them from the incubator and set up liquid cultures for you to use next time.

For next time

 * 1) Refer to the W/F section control digest gel image and answer the following questions, briefly explaining how you determined your answers.
 * 2) *Does the uncut backbone run primarily in nicked or in supercoiled form?
 * 3) *Which enzyme is a worse cutter, XmaI or BamHI?
 * 4) *Look up the NEB notes page for the poor cutter. What protocol is recommended for plasmid DNA?
 * 5) Some of you may have been surprised by the direction of activity increase for your Day 4 transfer function. Let's investigate why. Look back at the Day 1 exercises and the full pseudo-edge detector circuit (IPTG-sensitive). Re-draw the circuit with the communication module absent, and use it to explain your results.
 * 6) As you read the feedback on your Module 1 report, consider how the comments can be translated to your writing in Module 2. If you apply the lessons now, you may not need to revise your second report at all! Nevertheless, to give you some opportunity for feedback, you will draft a couple of parts in advance.
 * 7) *Before the first week of the module completely leaves your mind, prepare a schematic introducing your Plux-&lambda; design. Be sure to clearly indicate how it is different from the original. For now, briefly describe your approach in the caption. Later, that text can move to the main body of the report.
 * 8) **If you got no colonies (as indicated in my most recent email), you can make a schematic either for your original design, or for the new design you have chosen. Ultimately your report will need to include a schematic for the DNA you actually work with.
 * 9) *Write a general Methods sub-section for running &beta;-gal assays, including culture preparation. Eventually you will fill in all the different cell strains that you used.
 * 10) *Prepare a figure and caption for the IPTG:lacZ transfer function data.
 * 11) Please download the midsemester evaluation form [[Media:MidsemesterEval_20.109-S11.doc | found here]]. Complete the questionnaire and then print it out without including your name  to turn in. If there is something you'd like to see done differently for the rest of the course, this is your chance to lobby for that change. Similarly, if there is something you think the class has to keep doing, let us know that too. Your feedback is taken most seriously.

Reagent list

 * LB (Luria-Bertani broth)
 * 1% Tryptone
 * 0.5% Yeast Extract
 * 1% NaCl
 * autoclaved for sterility


 * Ampicillin: 100 mg/mL, aqueous, sterile-filtered


 * LB+AMP plates
 * LB with 2% agar and 100 &mu;g/ml Ampicillin


 * T4 DNA ligase buffer (1X), from NEB
 * 50 mM Tris-HCl
 * 10 mM MgCl2
 * 10 mM DTT
 * 1 mM ATP
 * 25 &mu;g/ml BSA


 * XL1-Blue competent cells