REVISE FROM S11 M2
Last time you were here, you transformed your new construct — pED-IPTG-YFD — into XL1-Blue cells. As you can see in the linked manual (PDF), these cells have the following genotype: recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F ́ proAB lacIqZΔM15 Tn10 (Tetr)]. Two gene mutations make XL1-Blue very useful "workhorse" cells for cloning. First, endA1 limits the non-specific destruction of plasmid (and chromosomal) DNA normally carried out by the EndA enzyme, thus maximizing DNA recovery. Second, recA1 makes the cells incapable of homologous recombination, which could otherwise cause undesirable intermingling between the plasmid and chromosomal DNA.
You will extract DNA from three independent colonies that we picked and grew in liquid culture overnight. The procedure you will do is commonly termed "mini-prep," which distinguishes it from a “maxi-” or “large scale-prep” that involves a larger volume of cells and additional steps of purification. The overall goal of each prep is the same--to separate the plasmid DNA from the chromosomal DNA and cellular debris, allowing the plasmid DNA to be studied further. In the traditional mini-prep protocol, the media is removed from the cells by centrifugation. The cells are resuspended in a solution that contains Tris to buffer the cells and EDTA to bind divalent cations in the lipid bilayer, thereby weakening the cell envelope. A solution of sodium hydroxide and sodium dodecyl sulfate (SDS) is then added. The base denatures the cell’s DNA, both chromosomal and plasmid, while the detergent dissolves the cellular proteins and lipids. The pH of the solution is returned to neutral by adding a mixture of acetic acid and potassium acetate. At neutral pH the SDS precipitates from solution, carrying with it the dissolved proteins and lipids. In addition, the DNA strands renature at neutral pH. The chromosomal DNA, which is much longer than the plasmid DNA, renatures as a tangle that gets trapped in the SDS precipitate. The plasmid DNA renatures normally and stays in solution, effectively separating plasmid DNA from the chromosomal DNA and the proteins and lipids of the cell.
Normally in 20.109 we do an in-house mini-prep procedure according to the steps above followed by ethanol precipitation. However, because we are working with a low-copy plasmid, today we will use a commercially available kit to give you the best chance of success. The principle is the same as that of our "quick and dirty" (and cheaper!) prep, but is combined with the silica gel column purification you are familiar with from using other Qiagen kits.
We discussed last time that not all of your colonies may be carrying the correct plasmid, which is why you will isolate three separate clones. The most likely incorrect plasmid is the original pED-IPTG-INS, one that was singly cut (or less likely not cut at all) during the enzymatic digestion. Part of your miniprepped DNA will be sent for sequencing, and you will analyze the results next time to determine a clone to move forward with. In the meantime, you will transform all three candidates clones into the edge detector cell strain, JW3367c.
In order to make our IPTG-sensitive results as comparable as possible to the light-based system, we are using the JW3367c strain even though strictly speaking we don't have to. The genotype of a precursor to this strain can be seen at this link. Of particular importance to the edge detection system are the deletions of native lacZ and envZ, which could otherwise cause background production of black pigment or cross-talk in the 2-component signaling system, respectively. (Recall that the edge detector strain carries a plasmid with Cph8, a fusion of EnvZ and Cph1 that makes the former sensitive to light instead of salt concentration.) The difference between our strain and the linked one is that the envZ deletion is no longer combined with Kanamycin resistance, because KanR is used to mark the phycocyanobilin plasmid instead. Before transforming JW3367c, you will have to make these cells competent to take up foreign DNA by treatment with calcium chloride.
In another week you will finally get to see the results of all your hard work, and know how your modification affected the model system!
You will figure out which clone to use by analyzing your sequencing data.
Normal bases versus chain-terminating bases
The invention of automated sequencing machines has made sequence determination a relatively fast and inexpensive endeavor. The method for sequencing DNA is not new but automation of the process is recent, developed in conjunction with the massive genome sequencing efforts of the 1990s. At the heart of sequencing reactions is chemistry worked out by Fred Sanger in the 1970s which uses dideoxynucleotides (see schematic above left). These chain-terminating bases can be added to a growing chain of DNA but cannot be further extended. Performing four reactions, each with a different chain-terminating base, generates fragments of different lengths ending at G, A, T, or C. The fragments, once separated by size, reflect the DNA’s sequence. In the “old days” (all of 15 years ago!) radioactive material was incorporated into the elongating DNA fragments so they could be visualized on X-ray film (image above center). More recently fluorescent dyes, one color linked to each dideoxy-base, have been used instead. The four colored fragments can be passed through capillaries to a computer that can read the output and trace the color intensities detected (image above right). Your sample was sequenced in this way on an ABI 3730 DNA Analyzer.
Analysis of sequence data is no small task. “Sequence gazing” can swallow hours of time with little or no results. There are also many web-based programs to decipher patterns. The nucleotide or its translated protein can be examined in this way. Thanks to the genome sequence information that is now available, a new verb, “to BLAST,” has been coined to describe the comparison of your own sequence to sequences from other organisms. BLAST is an acronym for Basic Local Alignment Search Tool, and can be accessed through the National Center for Biotechnology Information (NCBI) home page.
deciding whether to do Qiagen or homebrew kit
Qiagen: old text to revise below
- Pick up your three candidates cultures, growing in the test tubes labeled with your team colour. Label three eppendorf tubes to reflect your candidates (C1-3).
- Vortex the bacteria and pour ~1.5 mL of each candidate into an eppendorf tube.
- Balance the tubes in the microfuge, spin them for two minutes, and remove the supernatants with the vacuum aspirator.
- Pour another 1.5 mL of culture onto the pellet, and repeat the spin step.
- Resuspend the cell pellet in 250 μL buffer P1.
- Buffer P1 contains RNase so that we collect only our nucleic acid of interest, DNA.
- Add 250 μL of buffer P2 and mix by inversion until the suspension is a homogeneous blue colour. About 4-6 inversions of the tube should suffice.
- Buffer P2 contains sodium hydroxide for lysing.
- The blue colour comes from a special reagent that is not required for purification, but is simply used to check one's mixing technique.
- Add 350 μL buffer N3, and mix immediately by inversion until there is no blue colour (4-10 times).
- Buffer N3 contains acetic acid, which will cause the chromosomal DNA to messily precipitate; the faster you invert, the more homogeneous the precipitation will be.
- Buffer N3 also contains a chaotropic salt in preparation for the silica column purification.
- Centrifuge for 10 minutes at maximum speed. Note that you will be saving the supernatant after this step.
- Meanwhile, prepare 3 labeled QIAprep columns, one for each candidate clone.
- Transfer the entire supernatant to the column and centrifuge for 1 min.
- Wash with 0.5 mL PB, then separately with 0.75 mL PE, with each spin step 1 min long.
- After removing the PE, spin the mostly dry column for 1 more min.
- It is important to remove all traces of ethanol, as they may interfere with subsequent work with the DNA.
- Add 30 μL of EB to the top center of the column, wait 1 min, then spin 1 min to collect your DNA.
- EB is elution buffer, or 10 mM TrisCl (pH 8.5).
Part 2: Measure DNA concentration
Part 3: Prepare sequencing reactions
Part 4: Count colonies
Part 5: Sensitivity/specificity analysis for microsporidia primers
For next time
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