Team:NOVA LxPortugal/Experiments

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Arabidopsis thaliana germination assays

To evaluate the effect of spectinabilin on seed germination, we inoculated Arabidopsis thaliana seeds in three different types of plates: Murashige and Skoog (MS) medium; MS medium with DMSO and MS medium supplemented with spectinabilin. The seeds inoculated in MS medium plates represent a positive control of germination.

Since spectinabilin was solubilized and stored in DMSO, we wanted to assess its effect on germination of the seeds to avoid incorrect attribution of secondary effects of DMSO to spectinabilin. Therefore, four plates with 10, 20, 100, 200 and 400 µL of DMSO were inoculated.

According to Min-Jiao et al.1, the LC50 value (concentration that kills 50% of the test subjects) of spectinabilin against Bursaphelenchus. xylophilus is 0.84 µg/mL. Therefore, five different concentrations below and above the LC50 value were tested: 0.05; 0.1; 0.5; 1 and 2 µg/mL.

After the inoculation of the A. thaliana seeds in the different tested conditions, all the plates were incubated at 4 °C in a dark chamber for 3 days. This step is crucial to overcome seed dormancy. It is known that temperature and maternal plant photoperiod during seed development plays a relevant role in setting dormancy level and, consequently, germination timing2,3. Hence, this step allowed seed synchronization and triggering of the germination-related processes.

The following step consisted in an incubation at 20 °C with a 12 h/12 h photoperiod enabling both photosynthesis and chemosynthesis, which contribute to seed germination and growth. One week later, we evaluated the germination rate and phenotype present in the three types of media.

Promoters insertion into cloning vector pSEVA234

The final aim of our project is to build a plasmid that activates the expression of the spectinabilin production genes in the presence of alpha-pinene. So, our first experiment is to find alpha-pinene inducible promoters and validate their induction by the compound. To achieve this, we selected two different promoters: the alpha-pinene oxide lyase promoter (P1), an enzyme from the alpha-pinene degradation pathway known to be overexpressed in the presence of this compound from Pseudomonas rhodesiae CIP107491 - and the P2, a promoter associated with the enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase, an enzyme from the alpha-pinene catabolic pathway from Pseudomonas putida. To validate the efficiency of the promoters inducibility by alpha-pinene, two DNA constructs containing the Green Fluorescent Protein (GFP) under the control of each promoter and a terminator sequence downstream were built (P1_GFP_T and P2_GFP_T) and cloned into the plasmid pSEVA234, which is compatible with the P. putida strain. The goal was to infer if the supplementation of alpha-pinene could induce the expression of GFP and consequently fluorescence could be observed.

Two different molecular biology strategies were designed to clone each promoter: one eliminating the original operon lac and trc promoter from the plasmid pSEVA234, and another one cloning the DNA constructs directly in the original multiple cloning site of the plasmid. For the first strategy, the restriction enzymes used were PacI and KpnI, while for the last design, EcoRI and KpnI were the selected enzymes. In Figure 1, a scheme of the constructed plasmids is shown.

Figure 1 - Scheme of the constructed plasmids

The first step in this assay was to amplify both DNA inserts with the two different promoters (P1 and P2) and the terminator by Polymerase Chain Reaction (PCR). This technique allows the generation of copies of a specific DNA sequence in vitro, relying on a thermostable DNA polymerase, Taq polymerase. In a PCR reaction, it is necessary to add forward and reverse primers (short sequences of nucleotides flanking the target DNA sequence) that bind to the DNA template and can be extended by Taq polymerase with the addition of nucleotides.

The PCR protocol includes three main steps as represented in Figure 2: denaturation (at 98°C), in which the high temperature will denature DNA strands providing single-stranded DNA template; annealing (depending on the primers melting temperature), by lowering the reaction temperature the primer bind to their complementary sequences on the DNA template; extension (at 72°C), the step where Taq polymerase synthesizes new DNA strands through primers extension4.

Figure 2 - Scheme representing the main steps in Polymerase Chain Reaction main steps scheme. Created in BioRender

The genes used in this study were amplified by polymerase chain reaction (PCR) using Phusion High-Fidelity DNA Polymerase (Thermo Scientific) in a LifeECO Thermal Cycler (Bioer Technology). All DNA constructs and primers were purchased from IDT. DNA fragments were purified using DNA Clean and Concentrator DNA Kit from Zymo Research. The success of the PCR was checked by running an agarose gel electrophoresis. In Table 1, the sequence of the primers used in this project are summarized.

Table 1 - List of primers used in this study.
Primer Sequence (the restriction cloning sites are in italic) Restriction enzyme
P1_GFP_fw_PacI ccccccTTAATTAAcgatcgcccatgggc PacI
P2_GFP_fw_PacI ccccccTTAATTAAggctggggctgcaac PacI
P1_GFP_fw_EcoRI ccccccGAATTCcgatcgcccatgggc EcoRI
P2_GFP_fw_EcoRI ccccccGAATTCggctggggctgcaac EcoRI
P12_GFP_rev_KpnI ccccccGGTACCagcccccagtcgatg KpnI

Plasmids were extracted using NZYMiniprep kit (Nzytech). All digestions were performed using the appropriate FastDigest® restriction endonucleases (Thermo Scientific). Ligations were performed with T4 DNA Ligase (Thermo Scientific) and transformed by heat-shock in chemically competent E. coli DH5-alpha cells. The success of the ligation was checked through Colony PCR using DreamTaq (Thermo Scientific) and further confirmed by sequencing (StabVida). Protocols were performed in accordance with manufacturer’s instructions.


Arabidopsis thaliana germination assays

Murashige and Skoog (MS) medium

- 1x MS macro salts

- 10 g/L sucrose

- 0.5 g/L 2-(N-morpholino)ethanesulfonic acid (MES)

- 8 g/L agar

- Adjust pH to 5.7

MS medium + Dimethyl sulfoxide (DMSO)

- Four plates of MS medium with DMSO were made by adding 10, 20, 100, 200 and 400 µL of DMSO

MS medium + Spectinabilin

- Five plates of MS medium were supplemented with 0.05, 0.1, 0.5, 1 and 2 µg/mL of Spectinabilin

Arabidopsis seeds plate inoculation

  1. Place enough Arabidopsis thaliana seeds in 1.5 mL Eppendorfs to cover the bottom
  2. Add 200 µL of Ethanol 70%
  3. Vortex each Eppendorf for 20 to 30 seconds
  4. Remove the ethanol with caution to avoid seeds loss
  5. Add 200-250 µL of sterile H2Odd
  6. Vortex each Eppendorf for 10 to 20 seconds, followed by a spin-down to allow the seeds to seed
  7. Remove the water
  8. Repeat steps 5-7 three more times
  9. Add 200 µL of sterile H2Odd
  10. Using a pipette, place 10 seeds evenly over a line in the medium plate
  11. Repeat step 10 until each plate has approximately 40 isolated seeds
  12. Incubate the plates at 4°C, protected from light for 3 to 4 days to overcome seeds dormancy and to synchronize germination
  13. Change incubation conditions to 20°C with 12h/12h photoperiod for 1 week

Promoters insertion into cloning vector pSEVA234

  1. Amplification of P1 and P2 via Polymerase Chain Reaction (PCR)
  2. P1: promoter 1 – GFP – terminator (BBa_K3675004)

    P2: promoter 2 – GFP – terminator (BBa_K3675005)

  3. Purify both inserts with the DNA Clean and Concentrator Zymo Research Kit
  4. Digest both PCR inserts and plasmid pSEVA234 with the PacI/KpnI and EcoRI/KpnI
  5. Purify inserts and plasmid with the DNA Clean and Concentrator Zymo Research Kit
  6. Ligation of inserts with the plasmid using T4 Ligase
  7. Transform 5 μL of the ligation reaction into chemically competent E.coli DH5α cells by heat shock
  8. Plate the cells in LB-agar medium supplemented with kanamycin and grow overnight at 37 °C
  9. Colony PCR with appropriate primers to find successful clones
  10. Grow overnight positive colonies in LB+Kanamycin
  11. Plasmid extraction with NZYMiniprep kit and send for sequencing to confirm if the sequence is correct
  1. Liu, M.‐J., Hwang, B.‐S., Jin, C.‐Z., Li, W.‐J., Park, D.‐J., Seo, S.‐T. and Kim, C.‐J. (2019), Screening, isolation and evaluation of a nematicidal compound from actinomycetes against the pine wood nematode, Bursaphelenchus xylophilus. Pest. Manag. Sci., 75: 1585-1593. doi:10.1002/ps.5272
  2. Galloway LF, Etterson JR. (2007), Transgenerational plasticity is adaptive in the wild. Science 318: 1134–1136. doi: 10.1126/science.1148766
  3. Burghardt, L.T., Edwards, B.R. and Donohue, K. (2016), Multiple paths to similar germination behavior in Arabidopsis thaliana. New Phytol, 209: 1301-1312. doi:10.1111/nph.13685
  4. Garibyan L, Avashia N. Polymerase chain reaction. J Invest Dermatol. 2013;133(3):1-4. doi:10.1038/jid.2013.1