pUPD2 (Universal GB Part Domesticator):
The pUPD2 vector is a universal domesticator plasmid for level 0 parts. It is usually used in synthetic biology and for domestication of DNA parts for GoldenBraid assembly. It contains a chloramphenicol resistance gene and two cloning sites (BsmBI cutting sites), where in between there is a transcriptional unit from which β-galactosidase is expressed.
pDGB3α1 and pDGB3α2 vectors:
The pDGB3α vectors are universal domesticator plasmids for level α parts. They are usually used in synthetic biology and for domestication of DNA parts for GoldenBraid assembly. Each one contains a kanamycin resistance gene and two cloning sites (BsaI restriction sites), where in between there is a transcriptional unit from which β-galactosidase is expressed.
pDGB3ω vector:
The pDGB3ω vector is a universal domesticator plasmid for level ω parts. It is usually used in synthetic biology and for domestication of DNA parts for GoldenBraid assembly. It contains a spectinomycin resistance gene and two cloning sites (BsmBI restriction sites), where in between there is a transcriptional unit from which β-galactosidase is expressed.
1. Bacterial strains
DH5α Cells (Escherichia coli):
DH5α Competent Cells are Escherichia coli cells engineered to maximize transformation efficiency. They were used for cloning experiments.
GV3101 Cells (Agrobacterium tumefaciens):
GV3101 Agrobacterium tumefaciens cells were used for agroinfiltration experiments in Nicotiana benthamiana leaves. This strain contains the rifampicin resistant gene (rif) and the Ti-plasmid that carry the vir genes, which are necessary for the transformation of plant cells.
2. Nutrient media for bacterial cultures
LB Broth:
Luria Broth (LB) Broth was used in liquid form, dissolved in ddH2O and sterilized, for liquid bacterial cultures. The desired antibiotic should also be added.
LB Agar:
Luria Broth (LB) Agar was dissolved in ddH2O, sterilized and added in Petri dishes for bacterial cultures. The desired antibiotic should also be added before its solidification.
3. Antibiotics
The antibiotics used were the following: ampicillin, chloramphenicol, kanamycin, rifampicin and spectinomycin.
4. Enzymes
For the Restriction Enzyme Digestion and Ligation cloning experiments, the following enzymes were used: BsaI-HF, BsmBI, Esp3I and T4 Ligase.
For the Polymerase Chain Reaction, the Q5® High-Fidelity DNA Polymerase was used.
For the diagnostic digestion experiments, the following enzymes were used: BamHI-HF, BsaHI, BslI, EcoRI-HF, EcoRV-HF, HindIII-HF, NotI, RsaI and SspI.
5. PCR Reaction
Q5 High-Fidelity 2X Master Mix was used for all the PCR experiments.
6. DNA electrophoresis in agarose gel
TAE 50x electrophoresis buffer
The DNA electrophoresis buffer with a final volume of 1L contains 100 ml EDTA (0.5M), 242g Tris Base, 57.1 liquid acetic acid and ddH2O to final volume. The working solution is diluted from 50x to 1x.
Gel Loading Dye, Blue 6X
This loading solution consists of one tracking dye for agarose gel electrophoresis and contains SDS, which often results in sharper bands.
NEB 1kb DNA ladder molecular weight control
Aqueous solution of ethidium bromide (10mg/ml)
7. Reagents
Reagents for isolation of plasmid DNA from bacteria: “NucleoSpin® plasmid” kit from Macherey-Nagel
Ammonium molybdate
Ammonium molybdate reacts under acid conditions to form molybdophosphoric acid. In the presence of vanadium, yellow vanadomolybdo-phosphoric acid is formed, with the intensity of the yellow color being proportional to phosphate concentration. Absorbance is measured at 470 nm. Water from all sorts of samples can be used.
8. Agroinfiltration Experiments
MM buffer
MM buffer contains 10mM MgCl2 in 10mM MES buffer. The MES buffer is a common buffering agent, used in a lot of laboratory experiments as media, and MgCl2 is used to maintain cell homeostasis during leaf infiltration.
Acetosyringone
Acetosyringone is a naturally occurring phenolic compound secreted from wounded dicot plant tissues and is a chemoattractant for Agrobacterium. It is used to attract the bacteria to the plant area and to induce the vir genes, thus facilitating the transfer of the T-DNA region into the plant cells.
Hoechst 33342
Hoechst 33342 is a nucleic acid stain which emits blue fluorescence (Ex λ = 350 nm, Em λ = 461 nm) when bound to dsDNA. It was dissolved in PBS-tween and used for visualization of cell nuclei during the agroinfiltration samples’ observation in a confocal microscope.
9. Plants
Nicotiana benthamiana WT
Leaves of the model plant Nicotiana benthamiana were used for the agroinfiltration experiments.
Digestion-Ligation in one pot:
The Digestion-Ligation method is based on T4 Ligase’s ability to catalyze the formation of covalent phosphodiester linkages, which permanently attach complementary nucleotides. The ligation reaction is used to place a desired sequence (insert) in a plasmid vector. Usually, both of these sequences have previously been cut with the appropriate restriction enzymes, causing the formation of sticky ends, which can be designed to be compatible with the wanted row. T hese reactions can be catalyzed in repeated cycles to ensure a high efficiency.
| Reaction | 10 μL | Destination Vector | 50-75 ng (for pUPD2), 100-200 ng (for Alpha/Omega assemblies) |
| Part | 40-70 ng (for pUPD2), 200-400 ng (for Alpha/Omega assemblies) |
| 10X T4 DNA Ligase Buffer | 1 μL |
| T4 DNA Ligase | 1 μL |
| BsaI-HFv2 (Alpha clonings) or BsmBI-v2/Esp3I (L0 or Omega clonings) | 0.5 μL |
| ddH2O | x μL to reach 10 μL |
Use a thermocycler (PCR Machine) to run the following steps:
1. 5 min at 37℃ for BsaI/Esp3I or 42℃ for BsmBI
2. 5 min at 16℃
3. Repeat steps 1. and 2. 50-60 times
4. 10-20 min at 80℃
5. Rest at 16℃
Proliferation plasmid vectors from bacterial cultures
Competent E. coli bacteria preparation
Chemically competent cells are bacteria that have been treated with chemicals to enable them to insert exogenous DNA from their environment. For their p reparation, the yet incompetent cells are usually treated with salts like CaCl2 and MgCl2, whose role is the neutralization of the lipid membrane and the DNA, so as to allow the approach between them.
PROTOCOL
1. Take competent cells out of -80°C and thaw on ice
2. Mix 1-5μl of DNA into 100 μL of competent cells in a microcentrifuge or falcon tube. GENTLY mix by flicking the bottom of the tube with your finger a few times.
3. Incubate the competent cell-DNA mixture on ice for 20-30 min.
4. Heat shock each transformation tube by placing the bottom of the tube into a 42°C heatblock for 60 sec.
5. Put the tubes back on ice for 2-5 min.
6. Add 400 μl LB (without antibiotic) to the bacteria and grow at 37°C/210 rpm for 1h.
7. Plate the transformation reaction onto a LB agar plate containing the appropriate antibiotic and selection marker (40 μL X-gal, 10 μL IPTG for blue-white screening).
8. Incubate plates at 37°C overnight (at least 16h).
Verifying existence of plasmid vectors with the desired inserts
Small-scale isolation of plasmid DNA from bacterial cultures
To isolate and separate plasmid DNA from the rest of the cell's components and mainly from the "chromosomal" bacterial DNA, the alkaline lysis method was used. This method is based on the ability of plasmid DNA to rapidly reform into its double-stranded form, whereas larger DNA segments do not possess this ability.
PROTOCOL
From the bacterial colonies grown 2-3 were selected to grow overnight in culture in the presence of liquid LB medium with the appropriate antibiotic at 37℃ with agitation.
Alkaline lysis followed, which was performed with the NucleoSpin Plasmid Mini kit for plasmid DNA and according to the manufacturer's Isolation of high-copy plasmid DNA from E.coli protocol. The volume of elution solution used was 40μl.
Diagnostic digestion with restriction enzymes
Diagnostic digestion of the plasmid DNA isolated was performed to confirm the presence of the desired insert. Restriction endonucleases were used for this purpose.
PROTOCOL
1. Select restriction enzymes to digest your plasmid.
2. Determine an appropriate reaction buffer by reading the instructions for your enzyme.
3. In a 1.5mL tube combine the following:
| Reaction | 10 μL | DNA | 400-500 ng |
| 10x Buffer | 1 μL |
| Restriction Enzyme | 0,25 μL |
| dH2O | x μL (to bring total volume to 10µL) |
4. Mix gently by pipetting or flicking the bottom of the tube.
5. Incubate tube 37 °C for 1.5-2 hours.
DNA electrophoresis in agarose gel
Agarose gel electrophoresis is a technique by which DNA segments can be separated according to their molecular weight. The substance ethidium bromide (EtBr) is used to visualize the samples during electrophoresis. When conjugated to DNA, ethidium bromide fluoresces about 20 times more than its unconjugated form, thus making it possible to detect DNA segments in a UV table.
PROTOCOL
Sample preparation
| Plasmid | 1-2 μL | dH2O | 8-9 μL |
| Loading Dye | 2 μL |
1. Measure the appropriate volume of 1X TAE and 0,6gr of dry agarose.
2. Mix TAE and agarose in a flask and gently swirl
3. Microwave for 2-3 min (until it boils)
4. Mix it thoroughly under running water until it cools down.
5. When in the right temperature, add 2,16μl EtBr. Mix the flask to spread the EtBr everywhere.
6. Prepare the gel electrophoresis scaffold.
7. Add the liquid gel slowly into the gel box and let the gel solidify for 10-15 min.
8. Put the gel in the “electrode box”, filled with 1X TAE.
9. Load the samples in the gel wells.
10. Add the electrodes, set the gel electrophoresis instrument at 110V and run the gel for 30 min.
11. Put the gel under UV and take a photo to see what you did.
Integrity check of level 0 constructs
The plasmid vectors isolated were sent for sequencing to confirm the presence of the desired insert sequence. Sequencing was done at CeMIA SA (Larissa, Greece). The primers hybridize to the pUPD2 vector and are listed in the page PARTS of our wiki.
Integrity check of level 0 constructs
Agroinfiltration
Electrocompetent Agrobacterium cells preparation
Electrocompetent Agrobacterium cells are used for their ability to take up the desired plasmid via electroporation, with the end goal of transforming plants. They are prepared by washing them with ice-cold solutions, i.e. ddH2O and 10% glycerol solution, to remove any salts from the pellet suspension and, therefore, to prevent any arching during their transformation and to achieve a high transformation efficiency.
PROTOCOL
Day 1:
Take from glycerol stock a small quantity and add it to the pre-culture; 5ml LB with Rifampicin 20 μg/ml
Day 3 (Evening):
1. Inoculation of a 100mL sterile LB culture with cells from the pre-culture
2. Add appropriate volume to have a final OD600 of ~ 0.03-0.04
3. Incubate at 28℃ shaking incubator for 16h
Day 4:
1. Measure the OD600 of the cells to be 0.5-0.8. If it is bigger, dilute the culture again and re incubate it until it reaches 0.5-0.8. When you have the correct OD:
2. Place culture at 4℃ for 10-15 min to halt growth
3. Split the culture into two sterile 50 ml falcons
4. Centrifuge the falcons at 4000 x g for 10 min to harvest cells in 4℃
5. Discard the supernatant and resuspend cells with gentle swirls with ice-cold ddH2O (1X volume)
6. Repeat steps 3 and 4
7. Centrifuge the falcons at 4000 x g for 10 min at 4℃
8. Resuspend in ice-cold 10% glycerol (1/25 volume, i.e. 4 mL)
9. Centrifuge 4000 x g for 10 min and resuspend in ice-cold 10% glycerol (1/100 volume of the original culture, i.e. 1 mL or 1000 uL
10. Transfer 40 uL aliquots in 1.5 mL centrifuge tubes and snap-freeze in liquid nitrogen and store at -80℃
Agrobacterium tumefaciens Electroporation
Electroporation is a laboratory technique, used to introduce polar molecules, like DNA, to cells. This is done by applying electrical field to cells, which leads to increased cell membrane permeability.
PROTOCOL
1. Thaw the electro-competent aliquots on ice
2. Place clean electroporation cuvettes on ice
3. Mix 1-2 μL of DNA with your thawed cells, flick gently and put back on ice
4. Transfer the DNA-cells mixture from the tube into the respective pre-chilled cuvette
5. Use the Bio-Rad MicroPulser. Put the cuvette in the machine in the right orientation, move the cuvette towards the electrodes. Press the pulse button
6. Immediately add 900-950 μL LB in the cuvette and mix thoroughly. After mixing, transfer the transformed cells back to the tube you used to mix cells and DNA. Incubate at 28℃/160 rpm for 2-3 hours
7. Plate cells in LB Agar plates with correct antibiotics
Syringe Agroinfiltration in Nicotiana benthamiana leaves: 28℃
The method of agroinfiltration is used to transiently transform plant leaves. Nicotiana benthamiana leaves are usually preferred because of the ease of the technique, as it requires only a syringe and atmospheric pressure.
PROTOCOL
Day 1:
1. Preculture the agrobacterium strain in 5ml LB with appropriate antibiotics
2. 3 days incubation at 28℃
Day 4:
1. Inoculate 20 ml LB with 50μl of the preculture (with appropriate antibiotics)
2. 2 days incubation at 28℃ (shaking incubator)
Day 6:
1. Split the culture in 2x10ml in 50 ml falcons
2. 4.000 rpm for 10 min centrifugation
3. Remove supernatant carefully, wash the pellet with MM buffer -5ml in each falcon and resuspend GENTLY
4. 4.000 rpm for 10 min centrifugation
5. Remove supernatant and resuspend again pellet with MM buffer -5ml in each falcon
6. Gather them in 1 flask and add 200μM Acetosyringone (10μl)
7. Incubate at 28℃ at 100 rpm for 1-2h (dark)
8. Measure concentration in spectrophotometer with MM buffer as Blank. OD600 must be near 1
9. Find the correct dilution and then dilute the whole culture
10. This is now your inoculum! Put it in 2ml labeled eppendorf tubes and prepare small syringes
Infiltration
11. Turn the leaf upside down carefully and infiltrate the abaxial part of the leaf
12. First scar minimally with the syringe, then infiltrate the leaves avoiding the main veins of the leaves
13. Use a marker to circle around the infiltration points on the leaves to remember the infiltration sites and treatments
Observation
The working solution of Hoechst 33342 was prepared by mixing the powder into PBS-tween 1x with final concentration C = 10 ng/ml. The solution was, then, inserted to the N. benthamiana leaves, via negative pressure, for the nuclei observation. The samples were incubated for about an hour, in the dark.
After the incubation, the leaf samples were observed using confocal microscopy, with the following settings:
1. for the observation of mVenus Q69M: Ex λ = 515 nm, Em λ = 528 nm and
2. for the observation of Hoechst 33342: Ex λ = 350 nm, Em λ = 461 nm
Phosphorus Measurements
We used a common method for the colorimetric determination of phosphorus that is based on the reagents ammonium heptamolybdate and ammonium metavanadate (or molybdovanadate in short). This is a very simple method. Initially, a standard curve must be calculated for the calibration, with 10 different concentrations of KH2PO4 (10mM, 9mM, 8.4mM, 6mM, 4mM, 3mM, 2.1mM, 1mM, 0.5mM and 0.1mM). Then, the phosphorus-rich water samples reactions are prepared by adding vanadate-molybdate, shaking thoroughly and waiting 10 minutes for the color to develop fully. Absorbance is measured at 470 nm.
It is important for every scientist to record daily all the procedures followed in the laboratory. It is practical for personal use, to be able to understand the results of experiments, and also helpful for other scientists, who might want to replicate similar experiments. The Wet Lab members of our team understand the usefulness of such records and thus present you their Lab book.
Before an attempt is made to apply our device, it is necessary to construct it, characterize it and long-term test it. In this iGEM season, we achieved to build one of the two modules that constitute the final construct of our system, modified, and partially characterize it. Moreover, we managed to build the rest of the DNA pieces needed, but their right function and characterization require more experiments.
On this page, we present you the results of the experiments conducted during this summer with the aim of the above.
The aim of the following experiments was the construction of the transcriptional units mentioned below and their characterization in terms of their expression and intracellular localization.
We successfully built up different variations of the detection module of our system, with each one containing the Tet Repressor protein with several modifications (described in the Design and Engineering Success pages of our wiki). To be more specific, we designed the following variations:
Figure 1. Level α variants of Detection Module.
Constructs containing the mVenus fluorescent protein were designed and built with the aim to be characterized via confocal microscopy observation, while the rest were designed and built to be used as main parts of the final Detection module.
First, the DNA fragments, ordered from IDT and TWIST, were successfully inserted to the pUPD2 vector via GoldenBraid 2.0 Cloning System (see more in our Engineering page), which was confirmed by diagnostic restriction digestions (Figures 2 - 7).
Figure 2. Diagnostic Digestion of “mVenus for TetR” with RsaI. Expected bands (bp): 1212, 796, 538 and 282. The positive results are shown by the green arrows.
Figure 3. Diagnostic Digestion of: (2) “KRAB” uncut, (3) - (7) “KRAB” with EcoRI and HindIII (expected bands in bp: 2070, 417), (8) “TetR for KRAB & Venus” uncut, (9) – (10) “TetR for KRAB & Venus” with BsaHI (expected bands in bp: 2447, 304), (11) “TetR for Venus, no KRAB” uncut and (12) – (13) “TetR for Venus, no KRAB” with EcoRI and HindIII (expected bands in bp: 2070, 684) Positive results: (4) – (7), (9), (10) and (13) Negative results: (3) and (12) (1) MW
Figure 4. Diagnostic Digestion of: (7) “pNOS for TetR” uncut, (8) – (9) “pNOS for TetR” with EcoRI and EcoRV (expected bands in bp: 1506 and 896) (10) “Venus cds” uncut and (11) – (12) “Venus cds” with EcoRI and HindIII (expected bands in bp: 2070 and 756) Positive results: (7) – (12) (1) MW
Figure 5. Diagnostic Digestion of: (2), (3) “TPP riboswitch” with EcoRI and HindIII (expected bands in bp: 2473 and 308) (4), (5) “TetR for KRAB” with BsaHI (expected bands in bp: 2446 and 307) Positive results: (2) – (5) (1) MW
Figure 6. Diagnostic Digestion of “pNOS for Venus n TetR” with SspI and expected bands in bp: 1403 and 993. (1) MW, (2), (4), (6) and (8) digested (3), (5) and (7) uncut Positive results: (2), (4), (6) and (8)
Figure 7. Diagnostic Digestion of “TetR no KRAB” with EcoRI and HindIII and expected bands in bp: 2070 and 687. (1) MW, (2), (4) and (6) digested (3), (5) and (7) uncut Positive results: (2), (4) and (6)
Figure 8. Diagnostic Digestion of: (2) – (4) “pNOS-TetR-tNOS” with HindIII (desired bands in bp: 6345, 964 and 513) (5) – (7) “pNOS-Venus-tNOS” with HindIII (desired bands in bp: 6345, 1033 and 509) (2) – (4) “pNOS-Venus-TetR-tNOS” with HindIII (desired bands in bp: 6345, 1678 and 513) Positive results: all (1) MW
Figure 9. Diagnostic Digestion of: and (3) “pNOS-Venus-TetR-KRAB-tNOS“ with HindIII (expected bands in bp: 6345, 2056 and 513) and (5) “pNOS-TetR-KRAB-tNOS“ with HindIII (expected bands in bp: 6345, 1342 and 513) Positive results: (2), (4) and (5) (1) MW
Next, the level α constructs were inserted into Nicotiana benthamiana leaves via agroinfiltration experiments, to check their expression and their intracellular localization. The images taken during the observation of the samples are presented in Figures 10, 11 and 12. In this experiment, ddH2O was used as negative control, the construct pDGB3α2_pNOS-Venus-tNOS as positive control and Hoechst 33342 staining to observe the nuclei.
Figure 10. Agroinfiltration experiment: negative (ddH2O) and positive (pDGB3α2_pNOS-Venus-tNOS) controls.
In Figure 10, the signal of the negative control corresponds to the auto-fluorescence of the leaves and indeed it seems to appear only in circular structures that match the structure of chloroplasts. The positive control, on the other hand, emits a much stronger signal near the cell membranes. The presence of bridges, i.e. the fibers shown with red arrows, confirms that the protein is indeed being expressed in the cytoplasm and has simply been pressed towards the cell membranes due to the presence of large vacuoles, which is typical for leaf cells. It must also be mentioned that Hoechst 33342 staining efficiency was lower than expected, however for this part of our experiments it did not create significant hindrances.
Figure 11. Agroinfiltration experiment: pNOS-Venus-TetR-tNOS.
Moving on to the main constructs of the first module, the transcriptional unit containing the Venus-TetR protein was observed in circular structures that seemed to correspond to nuclei (red arrows, Figure 11), due to their shape but also their localization within the cell, as can be seen in the merged image. In addition, the chloroplasts, visible in this image, have a much lower signal and different size, similar to those of Figure 10 (negative control). To conclude, it is safe to say that pNOS-Venus-TetR-tNOS is mainly expressed in nuclei of leaf cells.
Figure 12. Agroinfiltration experiment: pNOS-Venus-TetR-KRAB-tNOS.
Last but not least, the construct pNOS-Venus-TetR-KRAB-tNOS was used to characterize the expression and localization of the protein Venus-TetR-KRAB. In Figure 12 a) and b), Hoechst 33342 staining efficiency was again low and thus was used to identify some nuclei where its signal was emitted more strongly. However,in both cases, where possible nuclei are observed, Venus-TetR-KRAB protein’s signal is also visible (red arrows), with higher intensity than the surrounding area. In Figure 12 c), a nucleus is present clearly, given its size and shape, the existence of which is unfortunately not confirmed by Hoechst 33342, which apparently did not work particularly well in this leaf area.
All the agroinfiltration results together, show that the TetR and TetR-KRAB proteins are expressed as expected, i.e. in the cell nucleus. Of course, more experiments like this must be conducted to confirm this conclusion.
Aiming the characterization of the expression and intracellular localization of the second module, we successfully constructed the following transcriptional units:
Figure 13. Level α variants of Nutrient Uptake Module.
As mentioned in the Design and Engineering pages of our wiki, we designed several variations of this module and used pht1 genes from different plant organisms with the purpose to compare their expression. All these variations would also be used as parts of the final system.
Once again, the first step was to import the DNA fragments, provided from IDT and TWIST, in pUPD2 vectors using the GoldenBraid 2.0 Cloning System (Figures 14 - 17).
Figure 14. Diagnostic Digestion of “TetO7” with EcoRI and EcoRV and expected bands in bp: 1189, 896 and 412. (1) MW Positive results: (5) and (6) Its sequence was also confirmed by sequencing.
Figure 15. Diagnostic Digestion of “Venus for PHT1” with EcoRI and HindIII and expected bands in bp: 2070 and 759. (1) MW, (14) uncut and (15) and (16) digested Positive results: (15) and (16)
Figure 16. Diagnostic Digestion of: (1) “tNOS-stuffer” uncut (2) and (3) “tNOS-stuffer” with BslI (desired bands in bp: 837, 785, 678, 279, 166 and 18) (5) “AtPHT1;5 opt” uncut (6) “AtPHT1;5 opt” with EcoRI (desired bands in bp: 2838 and 905) (13) “pTriple” uncut (14) and (15) “pTriple” with EcoRV (desired bands in bp: 1800 and 1398) Positive results: all (“tNOS-stuffer” was confirmed by sequencing) MW
Figure 17. Diagnostic Digestion of: (6) and (7) “OsPHT1;6 opt” with EcoRI and HindIII (desired bands in bp: 3387 and 332) (10) and (11) “AtPHT1;5 non” with EcoRI (desired bands in bp: 2254, 905 and 584) (12) “OsPHT1;6 part” with EcoRI and BamHI (desired bands in bp: 2310, 754 and 658) Positive results: all (1) MW
pDGB3α1_TetO7-pTriple-Venus-tNOS(s) contained the DNA fragment “Venus cds”, that was used for the 1st module, too.
Then, cloning experiments were conducted for the construction of the level α transcriptional units, mentioned above, using the pDGB3α1 destination vector. The success of these experiments is confirmed by the following Figures 18 and 19:
Figure 18. Diagnostic Digestion of “TetO7-pTriple-Venus-tNOS(s)” with EcoRI and HindIII and expected bands in bp: 6345, 1829, 674 and 387. (1) MW Positive result: (7)
Figure 19. Diagnostic Digestion of: (2) and (3) “TetO7-pTriple-AtPHT1;5 opt-Venus-tNOS(s)” with EcoRI and HindIII (desired bands in bp: 6345, 1978, 1492, 674 and 387) (4) and (5) “TetO7-pTriple-AtPHT1;5 non-Venus-tNOS(s)” with EcoRI and HindIII (desired bands in bp: 6345, 1978, 908, 674, 584 and 387) (6) and (7) “TetO7-pTriple-OsPHT1;6 opt-Venus-tNOS(s)” with EcoRI and HindIII (desired bands in bp: 6345, 2041, 1405, 674 and 387) (8) and (9) “TetO7-pTriple-OsPHT1;6 part-Venus-tNOS(s)” with EcoRI and HindIII (desired bands in bp: 6345, 3449, 674 and 387) Positive results: (2), (4) – (6) and (9) (1) MW
The level α constructs were inserted in Nicotiana benthamiana leaves with the aim of observing the proteins that would be produced via confocal microscopy. Unfortunately, the confocal microscope we were supposed to use output error indication and as a consequence, we did not manage to observe the leaves and get results.
However, we decided to use the Plate Reader to obtain fluorescence emission data from our samples. To be more specific, the leaf samples from the agroinfiltration experiment mentioned above, were cut into small pieces and one leaf piece from each sample was placed at the bottom of a well. The plate was then inserted to the Plate Reader with the following settings: Ex λ = 515 nm, Em λ = 528 nm. The results of these measurements are shown in the following diagram (Figure 20):
Figure 20. Agroinfiltration Experiment - Measurements of Plate Reader. GFP: leaf sample from N. benthamiana 16C and Negative Control: ddH2O.
From these results it appears that the positive control, i.e. pDGB3α1_TetO7-pTriple-Venus-tNOS(s), produces a higher signal than the negative control and thus that Venus protein is expressed at high levels. Unfortunately, the measurements for the PHT1-Venus constructs are similar to the negative control, which probably means that any fluorescence observed is probably due to the autofluorescence of the leaf.
Although this procedure is not standardized in our laboratory, we can make the following conclusions: (1) the TetO7-pTriple promoter works as expected and (2) the PHT1 constructs did not expressed properly, probably due to their complex protein structure in combination with the addition of another cds (Venus).
After successfully characterizing the two modules (detection and nutrient uptake modules), we aimed to combine them into one functional device. Unfortunately, the second module did not work as expected.
However, the final constructs containing both modules in various combinations, were made (Figures 22 and 23) with the purpose of testing our whole design. The destination vector used was the pDGB3ω1 and the constructs the following:
Figure 21. Level Ω variants of final construct.
Figure 22. Diagnostic Digestion of: (1) “TetO7-pTriple-Venus-tNOS(s)_pNOS-TetR-tNOS” with BamHI and HindIII (desired bands in bp: 6674, 2217, 1615 and 517) (2) “TetO7-pTriple-Venus-tNOS(s)_pNOS-TetR-KRAB-tNOS” with BamHI and HindIII (desired bands in bp: 6674, 2217, 1993 and 517) (3) MW (4) “TetO7-pTriple-AtPHT1;5 opt-Venus-tNOS(s)_pNOS-TetR-tNOS” with BamHI and HindIII (desired bands in bp: 6674, 3858, 1615 and 517) (5) “TetO7-pTriple-AtPHT1;5 opt-Venus-tNOS(s)_pNOS-TetR-KRAB-tNOS” with BamHI and HindIII (desired bands in bp: 6674, 3858, 1993 and 517) (6) “TetO7-pTriple-AtPHT1;5 non-Venus-tNOS(s)_pNOS-TetR-tNOS” with BamHI and HindIII (desired bands in bp: 6674, 3858, 1615 and 517) (7) “TetO7-pTriple-AtPHT1;5 non-Venus-tNOS(s)_pNOS-TetR-KRAB-tNOS” with BamHI and HindIII (desired bands in bp: 6674, 3858, 1993 and 517) (8) “TetO7-pTriple-OsPHT1;6 opt-Venus-tNOS(s)_pNOS-TetR-tNOS” with BamHI (desired bands in bp: 6674, 3482 and 2484) (9) “TetO7-pTriple-OsPHT1;6 opt-Venus-tNOS(s)_pNOS-TetR-KRAB-tNOS” with BamHI (desired bands in bp: 6674, 3860 and 2484) (10) “TetO7-pTriple-OsPHT1;6 part-Venus-tNOS(s)_pNOS-TetR-tNOS” with BamHI (desired bands in 35bp: 6674, 3482 and 2484) (11) “TetO7-pTriple-OsPHT1;6 part-Venus-tNOS(s)_pNOS-TetR-KRAB-tNOS” with BamHI (desired bands in bp: 6674, 3860 and 2484) Positive results: (4) – (6) and (8)
Figure 23. Diagnostic Digestion of: (1) “TetO7-pTriple-Venus-tNOS(s)_pNOS-TetR-tNOS” with BamHI and HindIII (desired bands in bp: 6674, 2217, 1615 and 517) (2) “TetO7-pTriple-Venus-tNOS(s)_pNOS-TetR-tNOS” uncut (3) “TetO7-pTriple-Venus-tNOS(s)_pNOS-TetR-KRAB-tNOS” with BamHI and HindIII (desired bands in bp: 6674, 2217, 1993 and 517) (4) “TetO7-pTriple-Venus-tNOS(s)_pNOS-TetR-KRAB-tNOS” uncut (5) “TetO7-pTriple-AtPHT1;5 opt-Venus-tNOS(s)_pNOS-TetR-KRAB-tNOS” with BamHI and HindIII (desired bands in bp: 6674, 3858, 1993 and 517) (6) “TetO7-pTriple-AtPHT1;5 opt-Venus-tNOS(s)_pNOS-TetR-KRAB-tNOS” uncut (7) MW (8) “TetO7-pTriple-OsPHT1;6 opt-Venus-tNOS(s)_pNOS-TetR-KRAB-tNOS” with BamHI and HindIII (desired bands in bp: 6674, 1993, 1793, 1350, 691 and 517) (9) “TetO7-pTriple-OsPHT1;6 opt-Venus-tNOS(s)_pNOS-TetR-KRAB-tNOS” uncut (10) “TetO7-pTriple-OsPHT1;6 part-Venus-tNOS(s)_pNOS-TetR-tNOS” with BamHI and HindIII (desired bands in bp: 6674, 2119, 1615, 964, 754 and 517) (11) “TetO7-pTriple-OsPHT1;6 part-Venus-tNOS(s)_pNOS-TetR-tNOS” uncut (12) “TetO7-pTriple-OsPHT1;6 part-Venus-tNOS(s)_pNOS-TetR-KRAB-tNOS” with BamHI and HindIII (desired bands in bp: 6674, 2119, 1993, 964, 754 and 517) (13) “TetO7-pTriple-OsPHT1;6 part-Venus-tNOS(s)_pNOS-TetR-KRAB-tNOS” uncut Positive results: (5) and (8) – (11)
We managed to build up seven of the ten level ω constructs and, unfortunately, there was no time to test neither of them in Nicotiana benthamiana leaves.
The last set of experiments that we conducted included the calibration step of the Molybdovanadate phosphate measurement method and the use of this method for the evaluation of our local river’s chemical state.
Figure 24. Different concentrations of K2HPO4, combined with the molybdovanadate reagent during the 10 minute waiting step.
This is the standard curve that was produced from the spectrophotometer measurements:
Figure 25. The absorbance is related to phosphorus concentration at a ratio of [P]/Abs = 7,2158.
In the end, we collected a sample from our local river and measured its absorbance (after the addition of molybdovanadate) at 0,025. This translates to a phosphorus concentration of 0,18 mM which is within the boundaries of literature data (Table 1).
| Phosphate levels at the Bridge of Larissa (Sample site) (mM) | |
|---|---|
| Minimum | 0.1 |
| Maximum | 7.4 |
| Mean | 3.2 |
Table 1. Phosphate levels in the Pineios river. Adapted from Mpoura N., (2015).
If we had successfully finished the experiments, mentioned above, and thoroughly characterized the expression of the final constructs, our next step would be the determination of their functionality. Thus, hairy root experiments, in the model plant Lotus japonicus, would take place, to observe the ability of the transformed roots to take up Pi at increased rates from their nutrient media.
After that, the responsiveness to microcystin-LR of the whole system would be checked. More specific, cloning experiments for the construction of variants of the Detection Module with several of the synthetic riboswitches (already designed, see more in our Engineering page) and agroinfiltration experiments in absence and presence of microcystin-LR in various concentrations would be conducted.
Thus, the functional riboswitches that would stand out would be included in the final constructs, which would have to be re-characterized with the experiments analyzed above (agroinfiltration and hairy root), in absence and presence of microcystin-LR.
The variants of the final construct that would work better would continue in subsequent transformation experiments that would more closely resemble the real conditions of our project, i.e. experiments on Phragmites australis, nutrient media with water from Lake Karla, etc.
Of course, many of the above steps would require increased levels of biosafety measures to be applied, to ensure the safety of the environment, the society and the lab workers.
References
- 1. Mpoura N. (2015). Study of the burden to the Pineios river caused by nutrients. Diploma thesis at the National and Kapodistrian University of Athens