NOTE: Check the buffers before use for salt precipitation. Redissolve any precipitate by warming to 37°C.
The reaction was incubated at 37 °C for 1 hour.
4.2.2 Transformation
- Put 5 µL of ligation reaction of plasmid DNA in competent cells
- Incubate for 30 mins on ice.
- Heat shock for 1 min at 42°C. Put back on ice for 2 mins.
- Add 800 µL of SOC to tubes. Incubate at 37°C for 1 hour.
- Plate 100 µL of the cells in 2X YT agar (25mL) plates, containing 100 mg/mL ampicillin 25 µL, 20 mg/mL X-gal 40 µL and 0.1M IPTG 12.5 µL .
- The reaction was incubated at 37 °C for 1 hour.
- Add 5 µL 2X Rapid Ligation Buffer and 1 µL T4 DNA Ligase (Promega). Incubate the reaction overnight at 4 °C
4.2.3 Plasmid extraction
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Inoculate a single colony of E. coli DH5-α into 5 mL SOB in test tube. Grow O/N at 37°C for 18 hours
-
Cell were collected using centrifuged at 12,000 rpm 1 min supernatant was discarded.
-
Add 200 µL solution I and RNaseA 2 µL to resuspend the pellet by vortex
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Add 200 µL solution II (100 µL 2% SDS and 100 µL 0.4N NaOH) invert the tube to mix thoroughly. The solution should become clear and viscous.
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Add 200 µL solution III invert the tube to mix thoroughly or until a precipitate forms.
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Centrifuge at 12,000 rpm 5 min 4 °C Transfer the supernatant to a microfuge tube and add 600 µL isopropanol. Incubate at -20°C for 10 minutes.
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Centrifuge at 10,000 rpm 10 min 4 °C supernatant was discarded
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Add 1,000 µL 70% ethanol and centrifuge at 10,000 rpm 10 min 4 °C
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Remove ethanol from the pellet by incubated at 37 °C 5 min
-
Add 30 µL DI water to the pellets
4.2.4 DNA fragment analysis using colony PCR
PCR Reaction
4.3 Electroporation
4.3.1 Agrobacterium competent cell
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Use a single colony for 5 mL LB broth culture for 2 day
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Freshly growing cell 200 µL to new 5 mL LB broth culture for overnight
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Cells were collected using centrifuged at 5200 rpm 3 min supernatant was discarded
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Add 1000 µL 10% (v/v) sterile glycerol to resuspend the cells and centrifuge <2 time>
-
Add 1000 µL 10% (v/v) sterile glycerol and resuspend
- Kept -20 ◦C for 1 hr.
- Kept in ice with water before use.
4.3.2 Electroporation
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Use 100 µL competent cell mix with 1-5 µg plasmid DNA (5 µL)
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This mixture was loaded into a chilled electroporation cuvette (gap = 2 mm) add placed into the cuvette holder
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The electroporation was used with the following parameters: 2.5-3 kV, 25 µF capacitance and 400 Ohm resistance. (400-800 Ohm)
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LB broth 1 mL was added immediately to the electroporation cuvette and transfer to 1.5 mL tube. Incubated at 27 ◦C for 1-3 hr. with rotating
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Electroporated cells was spread at LB plate with antibiotic for 2 day at 28 ◦C
4.3.3 DNA fragment analysis using colony PCR
Primer
PCR Reaction
5) Agroinfiltration
A single colony of Agrobacterium strains EHA 105 was grown in 5 mL of LB broth medium supplemented with 2.5 µL 50 µg/mL rifampicin, whereas a single colony of EHA105-1304 was grown in 5 mL of LB broth medium supplement with 2.5 µL 50 µg/mL rifampicin and 5 µL 50 µg/mL kanamycin. The cells were cultured at 28 ◦C and 250 rpm for 2 days. Then 1 mL of the Agrobacterium culture was refresh in 5 mL of new LB broth medium supplemented with the appropriate antibiotic as indicated above and incubated at 5600 rpm for 3 min and resuspened in deionized water with 100 µM acetosyringone (3,5-Dimethoxy-4-hydroxyaceto-phenone) to a final OD600 of 0.5 according to the methods of Yamin and Debener (2010)
The fully-opened flowers of Dendrobium Sonia ‘Earsakul’ were infected by pricking the center of the sepals and petals using a sterile syringe-needle, and infiltrated with 0.5 mL of Agrobacterium suspension using a 1-mL needleless syringe. The infiltrated flowers were incubated at 25 ◦C in the dark in microtube containing water and enclosed in a transparent, rectangular covered box. The samples were then assayed for GUS expression. Each experiment was repeated three times.
6) Histochemical GUS assay
The histochemical GUS assay was performed according to the procedures of Jefferson et al. (1987). The infiltrated petals and sepals were immersed in 10 mL GUS staining solution containing
0.5 mg/mL X-gluc
0.5 mg/mL X-gluc
0.5 mg/mL K4(Fe(CN)6)
0.5 mg/mL K3(Fe(CH)6)
10 mM/mL EDTA
100 mM/mL Na2PO4 (pH 7.0)
0.5% triton X-100
20% methanol
RESULTS
PART 1
RESULTS FROM PRIMER DESIGN:
From the database of Nif genes of A. brasilense, 9 primer pairs of 9 Nif genes was designed, namely NifH, NifE, NifN, NifX, NifA, NifQ, NifD, NifK, and NifY, following the below table and presented the number of base pairs of each primer and any characteristics. Moreover, NifD primer pairs was selected from other research to test together.
RESULT FROM PCR METHOD AND PCR PURIFICATION
Bacterial DNA was extracted and amplified Nif fragments according to primers using the PCR method. The PCR products were checked in 1% agarose gel electrophoresis. Lane 1 is 1Kb marker and Lane 2-11 are amplified Nif fragments arranged from NifH, NifE, NifN, NifX, NifA, NifQ, NifD, NifK, NifY, and NifD.
From all PCR products, some products were found that were amplified as a single band and correct size, while some appeared as many bands of products presented as non-specific bands. However, the correct bands also appeared. If in the PCR solution contained a single band, it was purified using a PCR solution Kit but if the PCR product showed multiple band, we chose the correct band and cut from gel for PCR purification. After that, all PCR fragments were run to check the size of Nif fragments again in 1% agarose gel electrophoresis.
RESULTS FROM WHITE-BLUE SCREENING OF PGEM IN E.COLI:
After transforming PCR Nif fragments into pGEM and screening with white-blue colony, the results were white colonies of the 8 Nif genes, namely NifH, NifE, NifN, NifX, NifQ, NifD, NifK and NifY, while NifA fragment was not successful because it was too difficult to insert the large fragment into pGEM, hence the process is successful. However, the size of the insert fragment was checked using EcoRI digestion to plasmid before sending it to sequencing.
RESULT FROM DNA SEQUENCE ALIGNMENT.
After sequencing, the program BLASTn was used on the NCBI database to check the identity and alignment of 2 sequences from T7 and SP6 end to confirm the correct sequences. Only 6 Nif sequences were complete because some could not find a start or stop codon of genes. Some examples of alignment are shown below.
RESULTS FROM PCAMBIA PLASMID COLONY PCR:
Based on the data, the transfer of Nif genes from A. brasilense to E.coli is complete, and the base pair corresponds with the location of the gene in the plasmid. This suggests that the gene transformation is complete. Based on the data collected from the electrolysis gel, the transformation of Nif genes (Nif K,H,Q,X,E,N) from A. brasilense to E.coli and then to Agrobacterium tumefaciens (Agrobacterium) was completed, as shown by the number of base pairs that correspond with the location of the gene in the plasmid. Since the pCAMBIA, the plasmid of Agrobacterium, has all the Nif Genes from A. brasilense, Agrobacterium can be used to transfer the genes into a plant via infection, thus turning the crop of interest into a crop that has the ability to fix nitrogen independently without depending on denitrifying and nitrogen-fixing bacteria. In conclusion, with the successful transformation of nitrogen-fixing genes from A. brasilense to Agrobacterium, Agrobacterium can be used as a way to turn plants into GMOs with ease.
Culture in LB agar, kanamycin, and X-Gal IPTG. When pGEM is inserted, E. coli does not have a kanamycin resistance gene, which meant that it would not be able to grow. However, if pCAMBIA was inserted, it would grow, as it would have had the kanamycin resistance gene. The blue-white screening technique was used in order to determine colonies with our inserts. Colonies with the insert would appear white after waiting for 2 days.
RESULTS FROM PCAMBIA PLASMID EXTRACTION FROM AGROBACTERIUM TRANSFORMATION:
After transforming pCAMBIA containing Nif genes into Agrobacterium, the transformation failed and we got a few colonies. Then the plasmid was extracted and checked using restriction enzyme digestion. The heat shock transformation was found to not be suitable for Agrobacterium transformation, thus a change in the transformation method was needed.
RESULTS FOR ELECTROPORATION:
The co-cultured and transformation pCAMBIA into Agrobacterium using electroporation was needed because this technique is more efficient and increases the permeability of the cell membrane which makes it possible for the plasmid to enter the Agrobacterium. The result was confirmed by colony PCR using specific primer for those genes. Only 2 colonies were presented for NifH and NifN genes. Finally, obtaining NifH gene from A. brasilense for further study
After performing colony PCR with the Nif primer, the electrophoresis gel results indicate that NifH has been successfully transferred into Agrobacterium, which is the only gene that showed a bar after performing gel electrophoresis. The base pair number of NifH also corresponds with the result from the gel electrophoresis, meaning that the transfer of the NifH gene was completed.
For the control group, 5 sepals were selected and left at 37oC overnight, then had stains removed with 70% ethanol to see the effect of ethanol on sepal leaves. Meanwhile, for the experimental setup, pCAMBIA was injected into 5 different sepals of the orchid Dendrobium Sonia and left overnight while maintained at a constant temperature of 37oC. After the application of 70% ethanol to the sepals to remove the stains on the orchid was done to make the GUS stain clearer for identification. By comparing the results of the experimental setup with the control, it can be identified that the GUS staining process is complete, since there are blue stains around the injected area. When the staining is blue, it can be concluded that the plasmid from Agrobacterium. is successfully transferred over to plant cells.
