Detection
Goal
Create a working sandwich Lateral-flow assay with our own aptamers for plant pathogens.
Results
We were able to prove that our preparation of the gold-nanoparticle(AuNP)-aptamer-conjugates was successful on one occasion. The successful conjugation of AuNPs with aptamers increases their effective size, reducing their running speed in the agarose gel. The control AuNPs run faster as their effective size is smaller.
Figure 1: 1. lane: originally citrate buffered AuNP-aptamer-conjugates; 2. lane: control AuNPs.
Future directions
Get devices like the AirJet dispenser, for easier dispensing of the control and test line, desiccators for a better drying process and the clamshell laminator to hold the single parts of the test in place, for an easier building process of the LFA.
Ampelpflanze
Goal
A reporter plant that changes color according to the surrounding stressors.
Results
Transformation of Arabidopsis thaliana with the control construct induced a change of the coloration of the whole plant. This proved that the RUBY gene cassette was functional and capable of inducing the synthesis of betalain.
Figure 2: Arabidopsis thaliana transformed with P35S_RUBY showing constitutive red coloring.
The seedlings grown from seeds of the transformed plants didn't exhibit their respective color.
Future directions
For the constitutively induced controls more transformations need to be done and more seedlings need to be screened. Additionally the stress inducible promoters still need to be extracted from gDNA and cloned in front of the reporter genes.
Proteam
Goal
Express and purify one or more target proteins, so they can be used as targets for SELEX in the future
Results
We isolated one of our target proteins from cDNA, and were able to get it into the expression vector pET-21a(+) via Gibson assembly. The correct integration was also confirmed via sequencing
Figure 3: DNA-gel showing the amplification of UGT74F2 in the preceding PCR. The band can be observed at roughly 1400 bp, just as expected from UGT74F2. PR1 didn't show any amplification. The ladder used is a GeneRulerTM 1kb DNA ladder from Thermo ScientificTM.
We then successfully expressed said protein (UGT74F2) in E. coli.
Figure 4: Coomassie-stained SDS gel of *E. coli* cell extract from before and after the induction with IPTG. After the induction there is a clear band at roughly 50 kDa, which is the size we expect UGT74F2 to have. The ladder is a PageRulerTM Plus Prestained protein ladder from ThermoScientificTM.
Future directions
To actually use the protein as a target for SELEX it has to be purified first. As we added a His-tag to the protein, a His-tag-purification would be the way to go here.
(Cell-)SELEX
Goal
Evolve aptamers for our test with the Cell-SELEX method.
Results
We were able to establish some parts of the Cell-SELEX process in our lab, for example the incubation of the library with the target cells and the digestion of double-stranded DNA into single stranded DNA. For confirmation, we used a 2 % TBE Agarose gel. If the digestion was successful, a different running speed through the gel would be expected. This can indeed be seen in the gel.
Figure 5: Electrophoresis for digestion verification Lanes from left to right: 1. ssDNA (Clean up after digestion) 2. dsDNA Reference (Probe 1) 3. ssDNA (no clean up after digestion) 4. 50 bp ladder.
Even though the ladder did not separate properly, both single-stranded DNA (ssDNA) samples travelled significantly faster than the double-stranded DNA (dsDNA) reference.
Furthermore, we experimentally tested the optimal centrifugal forces for our Pseudomonas syringae cultures with the help of a cell-counter. First, we did a reference probe to get an idea of our baseline cell count. Next we centrifuged at different g-forces (150/500/1000/1500 x g) and took the supernatant to compare it with our reference. The fewer cells were counted, the more cells were pelleted. Another factor that we took into consideration is the "debris", cell counts that have a different diameter than our target cells. Only the peak was taken into consideration.
Figure 6: The data generated by the Cell Counter.
With the cell count / ml combined with the cell / debris ratio, we identified 1000 x g as the optimal g-force to minimize dead cells that can compromise our experiments while having sufficient pelletization.
Figure 7: Cell Counter values
Figure 8: Cell Count to debris ratio
Future directions
Perform enough Cell-SELEX cycles, to evolve the aptamers that bind specifically to the cell surface of the plant pathogen Pseudomonas syringae.