TITLE: MOLECULAR ANALYSES
AUTHORS: Marco A. de Oliveira, Lilian H. Florentino, Thais T. Sales, Rayane N. Lima, Luciana R. C. Barros, Cintia G. Limia, Mariana S. M. Almeida, Maria L. Robledo, Leila M. G. Barros, Eduardo O. Melo, Daniela M. Bittencourt, Stevens K. Rehen, Martín H. Bonamino, Elibio Rech

[DESCRIPTION]
This protocol details the molecular analyses of assembly of a serine integrase-based platform for functional validation of genetic switch controllers in eukaryotic cells

[STEPS]
SECTION: Primer design ● Timing 1d
1. Select approximately 20 nucleotides both upstream and downstream of the core region of each att site formed in the reporter plasmid after recombination takes place.
 

SECTION: Primer design ● Timing 1d
2. Use an online oligo design tool to define the best forward primers annealing to promoter sequence and reverse primers annealing to terminator sequence present in the reporter plasmid.
SECTION: Primer design ● Timing 1d
3. Define oligo pairs to obtain two amplicons for each reporter plasmid. 
 
 The primers used in our studies are presented in Table 7.
 
 
 
SECTION: Target sequence amplification by PCR ● Timing 5h
4. Use a high-fidelity polymerase with non-template–dependent terminal transferase activity to insert a deoxyadenosine and the ends of generated amplicons.
 

SECTION: Target sequence amplification by PCR ● Timing 5h
5. Prepare a PCR mix for all reactions plus one (n+1) to account for pipetting errors. Include a negative control with water instead of DNA; positive control will require a previous synthesis of the expected recombined reporter plasmid.
SECTION: Target sequence amplification by PCR ● Timing 5h
6. Combine the reagents in the order shown below in Table 8, mix well by vortexing and spin briefly:

TABLE 8. PCR reaction mix components
 
 
 
SECTION: Target sequence amplification by PCR ● Timing 5h
7. Add 24.5 µL of the PCR mix to 0.2 mL PCR tubes..

SECTION: Target sequence amplification by PCR ● Timing 5h
8. To each respective tube, add 20 ngof template DNA and adjust the final volume to 25 µl if the DNA is too concentrated.
 

SECTION: Target sequence amplification by PCR ● Timing 5h
9. Gently pipette each sample up and down ten times to mix thoroughly. Place the PCR microtubes into a thermal cycler, and run the following program listed in Table 9 (volume = 25 μL).

TABLE 9. PCR cycling condition 
 
 
 

SECTION: Target sequence amplification by PCR ● Timing 5h
10. Resolve amplicons by electrophoresis in agarose gel following PCR. Run settings and gel density will depend on amplicon size according to the analyzed gene length and oligo pairs used.
 

SECTION: Amplicon purification ● Timing 2d
11. Excise the amplicon bands by cutting a square around them with the help of a scalpel on a UV light or blue light transilluminator.
 

SECTION: Amplicon purification ● Timing 2d
12. Proceed with amplicon purification using commercial DNA Clean-Up and Concentration kits, following the manufacturer’s recommendations.
SECTION: Amplicon purification ● Timing 2d
13. Clone purified amplicons in an entry vector to ensure high-quality sequencing results. Although specifics may vary depending on the plasmid, we recommend a molar ratio of 1:3 (vector to amplicon) and 1.5 U of T4 ligase in 5 µl reactions with   incubation at 16 °C.

SECTION: Amplicon purification ● Timing 2d
14. DH10b chemically competent cells were transformed with ligation products.
SECTION: Heatshock transformation of DH10b  chemically competent cell ● Timing 3d
15. Add 5 µL of the ligation reaction to 200 µL of cells thawed on ice.

SECTION: Heatshock transformation of DH10b  chemically competent cell ● Timing 3d
16. Incubate cells on ice for 30 min.

SECTION: Heatshock transformation of DH10b  chemically competent cell ● Timing 3d
17. Subject cells to heat shock at 42 °Cfor 45 s and return to ice for 2 min.

SECTION: Heatshock transformation of DH10b  chemically competent cell ● Timing 3d
18. Add 1 mL of LB or SOC medium.

SECTION: Heatshock transformation of DH10b  chemically competent cell ● Timing 3d
19. Incubate at 37 °C for 60 min and then plate different dilutions on LB plates with appropriate selecting agents. Incubate 60 min at 37 °C.

SECTION: Heatshock transformation of DH10b  chemically competent cell ● Timing 3d
20. Screening for positive transformants by colony PCR. A polymerase with less fidelity can be used in this step. Combine the reagents in the order listed in Table 10 below, mix well by vortexing and spin briefly:
TABLE 10. PCR reaction mix components for colony screening.
 
 
 
SECTION: Heatshock transformation of DH10b  chemically competent cell ● Timing 3d
21. Add 25 µL of the PCR mix to 0.2 mL PCR tubes.

SECTION: Heatshock transformation of DH10b  chemically competent cell ● Timing 3d
22. With a sterile toothpick or 200 µl pipetting tip, pick approximately 1/3 of each colony and add it to their respective tubes containing the PCR mix.
SECTION: Heatshock transformation of DH10b  chemically competent cell ● Timing 3d
23. Gently pipette each sample up and down ten times to mix thoroughly. Place the PCR microtubes into a thermal cycler, and run the following program listed in Table 11 (volume = 25 μL)

TABLE 11. PCR cycling condition for colony screening
 
 
 
SECTION: Heatshock transformation of DH10b  chemically competent cell ● Timing 3d
24. Resolve amplicons by electrophoresis in agarose gel following PCR.
SECTION: Heatshock transformation of DH10b  chemically competent cell ● Timing 3d
25. Select multiple confirmed clones to isolate plasmids using commercial kits following the manufacturer’s recommendations and have the purified plasmids sequenced.
 

SECTION: Heatshock transformation of DH10b  chemically competent cell ● Timing 3d
26. Analyze sequencing electropherograms and alignment to expected sequences to confirm proper DNA recombination by Integrase activity. 
 

TABLE 12. Troubleshooting for the molecular analyses stage