Experiment 4: Rapid
Subcloning by Cre-Lox Site-specific Recombination and Protein Expression in E.
coli.
Overview:
Each group will use a Cre-Lox-based system to subclone the coding sequence of the nimA gene into a bacterial expression vector derived from pET15b. The vectors we are using were published as part of “The Univector Plasmid Cloning System”, which was designed to facilitate construction of many different plasmid constructs to express the same protein. For example, in experimental molecular and cellular biology, one often wants to express a eukaryotic protein in bacteria to use as an antigen for making antibodies and in eukaryotic cells (insects or yeast) to produce post-translationally modified versions of the proteins. In addition, it is often useful to add multiple different epitopes (epitope-tag) the proteins when expressed in both bacteria and in eukaryotic cells. Because each expression plasmid and each type of tag requires specific cloning strategies, it can take a long time to generate the necessary constructs. The univector system allows one to clone an open reading frame into a single donor plasmid, and then use the Cre-Lox site-specific recombination system to move the open reading frame into each of many different expression constructs all in an afternoons work. In our lab, we’ll just use a single expression plasmid, derived from pET15b, which is used to express proteins that are tagged with the 6-His epitope. We’ll confirm that the correct clone has been made and then will transform the clone a strain of E. coli commonly used for protein expression (BL21 cells). You’ll express the protein in BL21 cells, isolated total protein from those cells, run an SDS-PAGE gel and then detect your recombinant protein by western blot analysis.
Rights to The Univector Plasmid cloning system are owned by Clonetech. They have modified and improved upon the Univector system and call their product the Creator Cloning System. In the original Univector system, an ORF is cloned into a single donor plasmid (pUNI10 in this case) just downstream of a LoxP sequence. The recombinant donor plasmid is mixed with a “host expression vector” (in this case, pHB3-HIS6) that contains a LoxP site just downstream from a regulatable promoter, and the Cre recombinase is added. LoxP is the sequence recognized by the Cre recombinase of bacteriophage, P1, which binds to two LoxP sites and catalyzes a recombination event within them. If there is a single LoxP site on each of two plasmids, as is true in this case, the recombination event results in formation of a single larger plasmid. Moreover, the recombination event places the ORF downstream of the regulatable promoter in the host expression vector. The Univector system as initially supplied to me included 26 host expression plasmids, seven for expression in E. coli, seven for expression in a baculovirus vector in insect cells, nine for expression in yeast, and three for expression in mammalian cells. In one afternoon, you could subclone the ORF from pUNI10 into all 26 host expression vectors, making 26 new recombinant DNA clones. This would take many months the old way, even for the most experienced molecular biologist. A similar strategy using different recombinases is marketed under the Gateway Technology system developed and patented by Invitrogen.
The host expression vector in this lab is a derivative of a standard E. coli expression vector called pET15b. This is one of many commercially available pET vectors, which have multiplied considerably after the initial publication of this expression system by Studier and Moffat in 1986. The pET system is designed to facilitate high level expression of any protein in E. coli, even if expression of the protein is toxic to E. coli. High level expression and the ability to repress expression of the target protein until desired are provided by the use of the bacteriophase T7 promoter. This promoter is not recognized by E. coli RNA polymerase, so it is possible to construct plasmids with toxic genes in front of the T7 promoter because they are not expressed. Introduction of these plasmids into strains with regulatable expression of the T7 polymerase then allow expression of the gene. The strain typically used is derivative of BL21(DE3), which contain the gene for T7 polymerase under control of the lacUV5 promoter on a lambda phage lysogen (DE3). T7 expression is very low in this strain, and introduction of the plasmid pLysS provides the T7 enzyme, lysosome, which inhibits the low T7 polymerase activity present in DE3 lysogens before induction by IPTG and also serves to help lyse E. coli during isolation of the protein.
Chronological
Protocol:
1. Perform the Cre-Lox reaction and transform the products into DH5α cells by electroporation. The TAs will provide you with pAS4-16 and pHB3-HIS6, each at 0.4 ug/ul; 10X reaction buffer. Cre-Lox reaction set up: 1 ul pAS4-16
1 ul pHB3-HIS6
5 ul 10X Reaction Buffer
42
ul ddH2O
49 ul subtotal
Take the reaction to the TAs, who will add 1 ul of Cre Recombinase to your mixture. Inculabate at 37 degrees for 30 minutes. Save your pAS4-16 and pHB3-HIS6 for control DNAs in step 3 below.
2. Transform DH5α electrocompetent cells with your Cre-Lox reaction as follows
a) Dilute 2 ul of the reaction with 18 ul sterile, ddH2O and ask the TAs for a tube of DH5α electrocompetent cells. Keep the diluted reaction and the cells on ice.
b) Add 2 ul of the diluted reaction to the cells and mix by pipetting.
c) Take your ice bucket and P-20 pipettor to the TAs by the electroporation unit in the back corner of the lab. The TAs will have a chilled electroporation chamber ready for you and will walk you through the process.
d) Add 1 ml of NZY+ or SOC medium to the electroporation chamber to collect all the cells and transfer the cells + medium to a culture tube provided by the TAs. Incubate the transformation mixture at 37 degrees shaking for 1 hour.
3. Make 1:10 dilution (25 ul transformation mix plus 225 ul medium) of the transformation mixture using NZY+ or SOC medium. Plate all of the 1:10 dilution, 250 ul of the undiluted transformation mixture, and the remainder of the transformation mixture on L-KAN plates. To plate “the remainder” of the transformation mixture, spin the remaining transformation mixture for 20 seconds in the microfuge, remove all but the last ~ 100 ul of medium, resuspend the pellet in the remaining medium by vortexing, and plate all of that on one plate. The TAs will inculbate your plates at 37 degrees overnight and then select 5 colonies and grow cultures for you to miniprep during the next lab period.
4. Miniprep 5 of your transformants using the Qiagen kit.
a) Transfer ~1.5 mls of each cell culture into labeled, 1.5 ml centrifuge tubes.
b) Harvest the bacterial cells by centrifugation in a the microcentrifuge at
full speed for 1 min at room temperature (15–25°C).
c) 1. Resuspend pelleted bacterial cells in 250 μl Buffer P1 containing
RNase.
d)
Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times. 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.
e)
Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the
tube 4–6 times. To avoid localized precipitation, mix the solution
thoroughly, immediately after addition of Buffer N3. Large culture volumes
(e.g. ≥5 ml) may require inverting up to 10 times and vigorous shaking.
The solution should become cloudy.
f)
Centrifuge for 10 min at full speed in a table-top microcentrifuge. A compact white pellet will form.
g) Apply the supernatants from step 4 to the
QIAprep spin column by pipetting. Label
the tops of the spin columns.
h) Centrifuge for 30–60 s at full
speed. Discard the flow-through.
i) Wash QIAprep spin column by
adding 0.75 ml Buffer PE and centrifuging for 30–60 s at high speed.
j) 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.
h) Place the QIAprep column in a clean 1.5 ml microcentrifuge tubes that have the lid cut off (use scissors). To elute DNA, add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min at high speed.
j) Transfer the DNA solution to a clean, labeled, 1.5 ml microcentrifuge tube and store on ice. NOTE: there is no EDTA in EB, so keep the DNA cold to minimize the effects of any contaminating nucleases.
5. Digest your mini-prepped plasmids with PstI and analyze the digest by gel electrophoresis to determine which of your clones have the structure predicted a by simple Cre-mediated recombination between LoxP sites in the donor and target plasmids (predict results of digests using Vector NTI as described in the Vector NTI Instruction Handout. Run digests of pAS4-16 and pHB3-HIS6 as controls. The TAs will provide you with 280 ul of a Master Mix for PstI digests. Distribute 35 ul of the mix to each of 7 tubes. Add 5 ul of each of your mini-prepped plasmids to each of the first 5 tubes. Add 1 ul of pAS4-16 and 4 ul of ddH2O to the sixth tube. Add 1 ul of pHB3-HIS6 plus 4 ul of ddH2O to the seventh tube. Incubate the digests at 37 degrees for an hour. Note: Remember to name and label your clone DNAs and store them in your box at -20 degrees.
6. Add 4 ul of 10X loading buffer to each digest. Run 15 ul of each digest plus MW markers on a gel for 30 to 45 minutes. Get photo of your gel and determine which of your clones has a PstI digest pattern predicted for the correct product of the Cre-Lox reaction. Choose one of the correct clones for transformation into BL21(DE3)pLysS (step 5).
7. Transform chemically competent BL21(DE3)pLysS cells with 1 ul of DNA from a clone with the correct PstI restriction pattern. The TAs will provide you with a culture tube containing 50 ul of thawed, chemically competent cells on ice and some SOC medium. Save the SOC medium for use also in step 6.
a) Add 1 μl of the DNA solution directly to the cells. Stir gently to mix.
b) Place the tubes on ice for 5 min.
c) Heat the tubes for exactly 30 s in a 42°C water bath; do not shake.
d) Place on ice for 2 min.
e) Add 250 μl of room temperature SOC
f) Incubate at 37 degrees with shaking for 1 hour.
8. Plate transformation on L-KAN plates. The TAs will
provide you with 3 L-
9. When you get to class, quickly take a 1 ml sample of the culture of your pAS4-16 plus pHB3-HIS6 clone and then induce expression of the NIMA::6HIS gene by adding IPTG. We’ll be using the protocol from page 33 of the Novagen pET System Manual. The TAs will have prepared 20 ml L-KAN cultures that are ready for sampling and inducing when you get to the lab at 2:00 and will give you a tube containing 400 mM IPTG (the inducer). Remove 1 ml of the culture for your “uninduced sample”, pellet the cells in a 1.5 ml tube and place on ice. Induce the culture by adding 20 ul of a 400 mM IPTG stock. Pellet the cells in your uninduced sample by spinning for 1 minute in a microfuge. Wash the cells by resuspending in 1.0 ml TE, re-pellet the cells, remove all the TE, resuspend the cells in 50 ul fresh TE, and store the pellet at -20 degrees in your box.
10. Collect and wash a cell sample at 4:30 PM before leaving. The TAs will collect one additional sample from each culture at 7 PM and provide it to you in the next lab period. The TAs will collect samples and induce the LacZ::6HIS control culture.
11. Prepare protein samples for SDS-PAGE. Thaw the samples using your hands to warm them, but place the samples on ice AS SOON AS THEY ARE THAWED. Add 100 ul of 2X SDS sample buffer supplied by the TAs. The sample will become viscous as the cells lyse. Use the 1ml syringe and needle provided to reduce the viscosity of the sample by drawing it into the syringe and pushing it out back into the tube. Continue this until the sample become relatively easy to pull into the syringe and the viscosity no longer changes.
12. Heat the samples for 3 minutes at 85 degrees to denature proteins and then load each sample (3 per group) plus a pre-stained MW marker plus a HIS-tagged MW marker on SDS-PAGE gels prepared by the TAs. The order of samples should be: HIS-tagged MW marker; uninduced; induced for 2.5 hours; induced for 5 hours; Pre-stained MW markers. Two groups should be able to load samples on one gel. The TAs will run a gel on their lacZ::6HIS control samples. Run the gel at 200 Volts for 40 minutes.
13. Transfer the protein from your gel to a PVDF membrane by following the instructions of the TAs. The TAs will stain the blots with Ponseau S to visualize total protein and then put them in blocking solution at 4 degrees until the next lab period. The TAs will provide you with a photo of the Ponseau S stain result. Determine whether you can detect induction of NIMA::6HIS on the Ponseau S stain.
14. Probe your blot with the anti-HIS antibody solution provided to you by the TAs. Incubate in anti-HIS antibody for 1 hour at room temperature.
15. Save the antibody solution by pouring it back into the original tube in which it was provided and wash the blot with the buffer provided (3, five minute washes following the TAs directions). Probe the blot with secondary antibody for 30 minutes at room temperature.
16. Wash 3 times as before. Detect the secondary antibody using the protocol from the first half of BIO 510 (or an alternate protocol supplied to you at the start of class). You will be provided with an image of your blot in the next lab period. Images of coomassie stained gel and antibody-probed blot of the lacZ::6HIS control samples from last year are on the web stie. Determine whether or not you can detect induction of NIMA::6HIS on the western blot. Compare your results with those of the group on your lab bench.
17. Record and Discuss Results
What are the predicted PstI restriction digest patterns of pAS4-16, pHB3-HIS6 new, and pAS4-16 plus pHB3-HIS6 new? Which of your clones had the predicted pattern for the desired product of the Cre reaction? Provide a hypothetical mechanism to account for clones that do not have the predicted restriction digest pattern. Was there evidence of expression of the NIMA::6HIS fusion product in your samples? In any group’s samples? If expression was not detected, provide a hypothesis to explain the result that is consistent with the other results obtained in this experiment.