Jun 07, 2024
- Marco A. de Oliveira1,2,
- Lilian H. Florentino1,2,3,
- Thais T. Sales1,2,3,
- Rayane N. Lima2,3,
- Luciana R. C. Barros4,
- Cintia G. Limia5,
- Mariana S. M. Almeida2,3,
- Maria L. Robledo5,
- Leila M. G. Barros2,3,
- Eduardo O. Melo2,3,
- Daniela M. Bittencourt2,3,
- Stevens K. Rehen6,7,
- Martín H. Bonamino8,9,
- Elibio Rech2,3
- 1Department of Cell Biology, Institute of Biological Science, University of Brasília, Brasília, Distrito Federal, Brazil;
- 2National Institute of Science and Technology in Synthetic Biology (INCT BioSyn), Brasília, Distrito Federal, Brazil;
- 3Embrapa Genetic Resources and Biotechnology, Brasília, Distrito Federal, Brazil;
- 4Center for Translational Research in Oncology, Instituto do Câncer do Estado de São Paulo, Hospital das Clínicas da Faculdade de Medicina de Universidade de São Paulo, São Paulo, São Paulo, Brazil;
- 5Molecular Carcinogenesis Program, Research Coordination, National Cancer Institute (INCA), Rio de Janeiro, Rio de Janeiro, Brazil;
- 6D’Or Institute for Research and Education (IDOR), Rio de Janeiro, Rio de Janeiro, Brazil;
- 7Institute of Biomedical Sciences, Federal University of Rio de Janeiro, Rio de Janeiro, Rio de Janeiro, Brazil;
- 8Cell and Gene Therapy Program, Research Coordination, National Cancer Institute (INCA), Rio de Janeiro, Rio de Janeiro, Brazil;
- 9Vice-Presidency of Research and Biological Collections (VPPCB), FIOCRUZ – Oswaldo Cruz Foundation Institute, Rio de Janeiro, Rio de Janeiro, Brazil
- Elibio Rech: corresponding author: elibio.rech@embrapa.br;
Protocol Citation: 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 2024. MOLECULAR ANALYSES. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkoyzdv5r/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 20, 2023
Last Modified: June 07, 2024
Protocol Integer ID: 92697
Abstract
This protocol details the molecular analyses of assembly of a serine integrase-based platform for functional validation of genetic switch controllers in eukaryotic cells
Attachments
pbt9ca8tp.docx
83KB
Primer design ● Timing 1d
Primer design ● Timing 1d
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.
Note
▲CRITICAL STEP forward primer must anneal to attL, while the reverse primer will anneal to the attR sequence.
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.
Define oligo pairs to obtain two amplicons for each reporter plasmid.
Note
▲CRITICAL STEP: Primer pairs must consist of a forward oligo annealing to the attL site and a reverse oligo annealing to the terminator region for sequencing proper attR site formation in amplicon I and a forward oligo annealing to the promoter region and a reverse oligo annealing to the attR site for sequencing proper attL site formation in amplicon II. (Figure 5).
The primers used in our studies are presented in Table 7.
| A | B | C | D | |
| Oligonucleotides used for amplification of Amplicon I and sequencing of attL sites | ||||
| Promoter | Forward primer (5’ -> 3’) | nt | Model | |
| EFa_966F | TTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTG | 35 | Mammal | |
| 35S_282F | ATTGATGTGATATCTCCACTGACGTAAGGGATGACGCAC | 39 | Plant | |
| attR | Reverse primer (5’-> 3’) | nt | Model | |
| attR _Int2_R | GTGTCTACGCGAGATTCTCGCCGGACCGTCGACATACTGC | 40 | All models used | |
| attR _Int4_R | AGTTTTCAACCCTTGATTTGAATAAGACTGCTGCTTGTGT | 40 | ||
| attR _Int5_R | ATAACTCTCCTGGGAGCGCTACACGCTGTGGCTG | 34 | ||
| attR _Int7_R | CTGTGTGAGAGTTAAGTTTACATGGGCAAAGTTGATGAC | 39 | ||
| attR _Int9_R | TGGAAGTGTGTATCAGGTAACTGGATACCTCATC | 34 | ||
| attR _Int13_R | GTAGAACTTGACCAGTTGGTCCTGTAAATATAAGCAATCC | 40 | ||
| attR _phiC_R | CCAACTGGGGTAACCTTTGGGCTCC | 25 | ||
| attR _Bxb1_R | CTGGTCAACCACCGCGGTCTCCGTCGTCAGGATC | 34 | ||
| Oligonucleotides used for amplification of Amplicon II and sequencing of attR sites | ||||
| attL | Forward primer (5’-> 3’) | nt | Model | |
| attL_Int2_F | GGAGTAGCTCTTCGCCCGAGAACTTCTGCAAG | 32 | All models used | |
| attL_Int4_F | CGACCTGAAATTTGAATTAGCGGTCAAATAATTTGTA | 37 | ||
| attL_Int5_F | GACGGCCTGGGAGCGTTGACAACTTGCGCACC | 32 | ||
| attL_Int7_F | GTCCGTCTGGGTCAGTTGCCTAACCTTAACTTTTAC | 36 | ||
| attL_Int9_F | ATAATTGGCGAACGAGGTATCTGCATAGTTATTCCGAAC | 39 | ||
| attL_Int13_F | TCCAGATCCAGTTGTTTTAGTAACATAAATACA | 33 | ||
| attL_phiC_F | TGCCAGGGCGTGCCCTTGAGTTCTCTCAGT | 30 | ||
| attL_Bxb1_F | TGTCGACGACGGCGGTCTCAGTGGTGTACGGT | 32 | ||
| Terminator | Reverse primer (5’ -> 3’) | nt | Model | |
| TermiAni_205R | AATGATTTGCCCTCCCATATGTCCTTCCGAGTG | 33 | Mammal | |
| NOSt_283R | ATAACAATTTCACACAGGAAACAGCTATGACATGATTACG | 40 | Plant | |
Target sequence amplification by PCR ● Timing 5h
Target sequence amplification by PCR ● Timing 5h
Use a high-fidelity polymerase with non-template–dependent terminal transferase activity to insert a deoxyadenosine and the ends of generated amplicons.
Note
▲CRITICAL STEP Amplicon modification is important for cloning into pGEM-t-Easy to be sequenced.
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.
Combine the reagents in the order shown below in Table 8, mix well by vortexing and spin briefly:
TABLE 8. PCR reaction mix components
| A | B | |
| Component | Volume to add (µl) | |
| dH2O nuclease free | 18.65 | |
| Buffer 10x | 2.5 | |
| MgCl2 [50 mM] | 1.5 | |
| PCR Fw primer [10 μM] | 0.75 | |
| PCR Rev primer [10 μM] | 0.75 | |
| dNTP [10 mM] | 0.75 | |
| Taq DNA polymerase | 0.1 |
Add 24.5 µL of the PCR mix to 0.2 mL PCR tubes..
To each respective tube, add 20 ng of template DNA and adjust the final volume to 25 µl if the DNA is too concentrated.
Note
Negative controls were prepared first by adding an equivalent volume of nuclease-free water and closing lids before pipetting templates to minimize contamination risk.
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
| A | B | C | D | |
| Cycle no. | Denature | Anneal | Extend | |
| 1 | 94°C, 3min | |||
| 2-34 | 94°C, 30s | 65°C, 30s | 72°C, 60s | |
| 35 | 72°C, 5min |
Note
▲CRITICAL STEP Given the need for primers to align to a defined att site sequence, some parameter adjustments, such as Tm, GC content and 3’ end base composition, will be limited and can vary from one integrase reporter to another, requiring adjustments to PCR cycling conditions.
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.
Note
▲CRITICAL STEP Load the same negative control in the every gel both technical (PCR without DNA) and biological (PCR using DNA from groups transformed with only either reporter plasmid or integrase plasmid) to ensure obtained bands indicate DNA inversion by Integrase activity. ? TROUBLESHOOTING
Amplicon purification ● Timing 2d
Amplicon purification ● Timing 2d
Excise the amplicon bands by cutting a square around them with the help of a scalpel on a UV light or blue light transilluminator.
Note
▲CRITICAL STEP Use different scalpel blades for each band to avoid cross-contamination of samples. ! CAUTION UV light can damage DNA, nicking and possibly removing DNA strand ends and interfering with downstream cloning steps. When available, blue light is highly recommended. If using UV, proceed quickly, turning the transilluminator off after making the cuts in the gel.
Proceed with amplicon purification using commercial DNA Clean-Up and Concentration kits, following the manufacturer’s recommendations.
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 Overnight incubation at 16 °C .
DH10b chemically competent cells were transformed with ligation products.
Heatshock transformation of DH10b chemically competent cell ● Timing 3d
Heatshock transformation of DH10b chemically competent cell ● Timing 3d
2h 32m 45s
Add 5 µL of the ligation reaction to 200 µL of cells thawed On ice .
Incubate cells On ice for 00:30:00 .
30m
Subject cells to heat shock at 42 °C for 00:00:45 and return to ice for 00:02:00 .
2m 45s
Add 1 mL of LB or SOC medium.
Incubate at 37 °C for 01:00:00 and then plate different dilutions on LB plates with appropriate selecting agents. Incubate Overnight at 37 °C .
2h
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.
| A | B | |
| Component | Volume to add (µl) | |
| dH2O nuclease free | 18.65 | |
| Buffer 10x | 2.5 | |
| MgCl2 [50 mM] | 1.5 | |
| PCR Fw primer [10 μM] | 0.75 | |
| PCR Rev primer [10 μM] | 0.75 | |
| dNTP [10 mM] | 0.75 | |
| Taq DNA polymerase | 0.1 |
Add 25 µL of the PCR mix to 0.2 mL PCR tubes.
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.
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
| Cycle no. | Denature | Anneal | Extend | |
| 1 | 94°C, 10min | |||
| 2-34 | 94°C, 30s | 60°C, 30s | 72°C, 90s | |
| 35 | 72°C, 5min |
Resolve amplicons by electrophoresis in agarose gel following PCR.
Select multiple confirmed clones to isolate plasmids using commercial kits following the manufacturer’s recommendations and have the purified plasmids sequenced.
Note
▲CRITICAL STEP Have your samples sequenced in both directions and in replicates to check for sequencing errors and identify possible mutations and DNA damage resulting from integrase activity.
Analyze sequencing electropherograms and alignment to expected sequences to confirm proper DNA recombination by Integrase activity.
Note
Troubleshooting
TABLE 12. Troubleshooting for the molecular analyses stage
| A | B | C | D | |
| Step | Problem | Possible reason | Solution | |
| 10 | Unspecific amplification and unexpected bands on agarose gel | Oligos annealing at att sites has a 3’ end complementarity to both original and recombined att sites | Increase the annealing temperature to more sselective conditions |