Week 3: TOPO-based DNA Cloning of Nucleotide Binding Site (NBS) Sequences; Bacterial Transformation

Introduction

After obtaining the PCR-amplified NBS-containing sequence from the last lab exercise, the DNA fragment(s) must be cloned into a cloning vector and propagated in bacteria before DNA sequencing because, unlike standard PCR, degenerate PCR can produce more than one PCR product(s). Meanwhile, dideoxy sequencing can only sequence identical DNAs. Thus, we need to clone the PCR product(s) to obtain a single DNA clone for outsourcing sequencing. The StrataClone PCR cloning method is a modified version of TOPO TA cloning, a 5-minute, one-step cloning strategy for the direct insertion of Taq polymerase–amplified PCR products into a plasmid vector with topoisomerase. Its linear cloning vector contains the sequence 5′-(C/T)CCTT-3′ (loxP site) at both 5′ ends and topoisomerase I covalently attached to the free 3′ ends (Figure 1-6).¹⁵ Each topoisomerase I-attached end also has a modified uridine (U*) overhang, which can anneal to the extra 3′ Adenine residue (A) of the PCR product. This occurs because the non-proofreading Taq DNA polymerase adds a 3′-A to the PCR product without a template during PCR amplification. If a proofreading DNA polymerase is used, it is necessary to add a 3′-A overhang after completing PCR. Taq-amplified PCR products efficiently anneal to these vector arms through A-U base-pairing followed by topoisomerase I-mediated strand ligation. The resulting linear recombinant molecule, vector arm (ori)–PCR product–vector arm (amp/kan), is then transformed into a competent cell line expressing cre recombinase. The bacterial cre recombinase mediates recombination between two loxP sites at both ends of the linear recombinant DNA to form a circular DNA molecule, pSC-A-amp/kan (Figure 1-6).¹⁵ This circular recombinant DNA can replicate in bacteria cells grown on ampicillin or kanamycin media. The pSC-A-amp/kan vector also contains a lacZ′α-complementation cassette for blue-white screening.

Before cloning the PCR product into pSC-A-amp/kan, it is necessary to verify the size of the PCR product by agarose gel electrophoresis. If multiple bands appear after gel electrophoresis, the product with the expected size must be isolated from the gel before cloning. The amount of PCR product used for reaction is described by the manufacturer. In addition, if the PCR product is < 3 kb with a robust and specific amplification, the PCR product should be diluted at 1:10; for larger or poorly amplified fragments, the dilution is unnecessary. The PCR product is not diluted as long as ≤ 2 µl of PCR product is used. In this lab exercise, the degenerate PCR product yield is not as robust as standard PCR, and thus it is not necessary to dilute the PCR product.

After inserting the DNA into a vector, the next step is to transform the recombinant DNA into a bacterial host to propagate it. The common cloning bacteria, E. coli, is gram-negative and cannot naturally take up foreign molecules from the environment. In addition to electroporation, which uses a high electrical pulse to create pores on bacterial membranes to facilitate DNA entry, cold calcium chloride (CaCl₂) transformation is another common technique used for DNA cloning in prokaryotic cells, i.e., bacteria. Adding cold CaCl₂ to the cell suspension crystalizes cell membranes to form channels and binds DNA to lipopolysaccharides (LPS) on the surface of the bacterial wall because the positively charged calcium ions attract both the negatively charged DNA backbone and the negatively charged groups in the lipopolysaccharide (LPS). The DNA can then enter the cell upon a short pulse of heat when the chilled cells (~4°C) are heated to a higher temperature (42°C) for 45 sec. These steps facilitate the entry of cloned DNA into bacteria, which then propagate the cloned DNA.

For this week’s lab exercise, we will clone the PCR-amplified DNA fragment(s) from the last lab into the pSC-A-amp/kan cloning vector. The vector with the inserted DNA is then transformed into E. coli competent cells expressing cre recombinase by the cold CaCl₂ method, followed by Blue-White colony screening.

Procedure

Ligation and Bacterial Transformation

Protocols adapted from “StrataClone PCR Cloning Kit: Instruction Manual.”¹⁵

1. Prepare the ligation reaction mixture by combining (in order): 3 µl StrataClone Cloning Buffer; 2 µl of PCR product (5–50 ng) or a 1:10 dilution of a robust PCR reaction, and 1 µl StrataClone Vector Mix.
2. Mix the ligation reaction mixture gently by repeated pipetting, incubate the mixture at room temperature for 5 minutes, and then place the mixture on ice. Note: The cloning reaction may be stored at -20°C for later processing.
3. Retrieve and thaw one tube of 50 µl StrataClone SoloPack competent bacterial cells. To maintain the bacteria’s competency, put it on ice immediately after thawing.
4. Add 2 µl of the ligation reaction mixture to the tube of thawed competent cells and mix gently by flicking the tube several times (no pipetting).
5. Incubate the transformation mixture on ice for 20 minutes. During the incubation period, pre-warm LB medium to 37°C.
6. Heat-shock the transformation mixture at 42°C for 45 seconds and then immediately put the mixture on ice for 2 minutes.
7. Add 250 µl of pre-warmed LB medium to the transformation reaction mixture. Allow the competent cells to recover for ≥ 1 hour at 37°C with agitation. (Lay the tube on the shaker horizontally for better aeration.)
8. For blue-white color screening, prepare LB-amp/X-gal and LB/X-gal plates by spreading 40 µl of 2% X-gal on each plate.
9. After ≥ 1 hr incubation (step 7), plate 100 µl of 10^-6, 10^-5, and 10^-4 diluted transformation mixture in LB on the plates with no ampicillin (Amp^–/X-gal). When spreading < 50 µl of transformation mixture, pipette the cells into a 50-µl pool of LB medium on the plate before spreading.
10. Plate 50, 75, and 150 µl of the transformation mixture on the LB-Amp⁺/X-gal plates. Incubate all the plates upside down overnight at 37°C.
11. The next day, seal the plates with parafilm strips and put them upside down in the refrigerator in the afternoon. (Never leave your plates for another overnight.)
12. Count the number of white/light blue versus blue colonies on each plate and fill in the table below before coming to the lab next week. For the dilution plates, pick one with ~300 colonies (the most representative plate).

Note: Some recombinant colonies may appear light blue instead of white after prolonged incubation. Suppose the insert contains an in-frame start codon proximal to a ribosome binding site; a functional lacZ′-fragment fusion protein may be produced, which typically results in blue or light blue colonies for one insert orientation.

Data Analysis and Discussion

1. Make a flow chart of the ligation/transformation experiment (Steps 1–10). If you do not know what a flow chart looks like, please search the internet for examples.
2. a.   What is the bacterial concentration of the 50 µl frozen competent tube given by the instructor? For this calculation, you need to count the total number of colonies on one of the dilution plates without ampicillin by picking the most representative plate with ~300 colonies.
   2. What color(s) of bacterial colonies would you expect to see on these plates? Indicate in each kind of bacteria if it contains plasmid, with or without the insert.
   3. Why did/didn’t you see the expected results on these plates? Explain.
3. The amplified DNA by standard PCR can be sequenced directly without being cloned. Why couldn’t we send out our PCR product for sequencing without cloning?
4. a.   After transformation, what color(s) of the bacterial colonies did you see on the Amp⁺/X-gal plates? Indicate in each colony type if it contains plasmid with or without the insert.
   2. Calculate the % of colonies with the DNA insertion (dividing the number of recombinant colonies by the total number of colonies on each of the Amp⁺/X-Gal plates).
   3. Based on the strategy of TOPO cloning, are your results, as indicated in answer (b), expected? Explain.
5. As part of the homework, please watch the two videos before the next lab: “Monarch Plasmid Miniprep Kit Protocols”¹⁶ and “Tips for using the Monarch Plasmid Miniprep Kit.”¹⁷