Engineering Design Cycle
- Our experimental design follows the engineering design cycle described in the flowchart to the right:
- Research: We began the project by extensively searching the available primary literature on regulation of biosynthetic gene clusters in filamentous fungi. From this, we began to understand the complexity of these biological circuits, and notice the lack of available parts/information. We also discovered that Aspergillus is a common organism for BGC research.
- Imagine: With no labs available to us this year, imagination was key. We chose to design this procedure exactly how we would if our lab was open. This means we thought about biosafety limitations, available equipment, cost, potential setbacks, time constraints, etc. The head of our lab, Dr. Faciotti helped us to keep thinking realistically about all of these factors.
- Design: Our genetic constructs, which mark the final physical step we are able to accomplish in the design cycle, were designed meticulously based on real parts from real, available, reasonably-priced plasmids that were found in literature.
- Build: This step requires culturing of the organism, Aspergillus niger. We’ve dedicated a comprehensive, reproducible procedural handbook to the public, that shows our methods for accomplishing this. The other requirement is the synthesis of the genetic constructs we designed previously.
- Test: To test the functionality of the binding sites in our constructs, we transform our cultured Aspergillus niger cells with our genetic constructs. We do this for each binding site, and look for a change in expression of our reporter gene, to signify functionality.
- Learn: Ideally, we would have tangible results in our hands to learn from, and re-test based on our observations. Instead, we tried to preemptively consider potential setbacks, and discuss our solutions to them in this experimental design.
- Improve: While it is difficult to talk about improvement before getting any real results, we have discussed many improvements to our computational methods. Memescape allows us to optimize parameters. We also have a backup method (which we did not need to use) for finding orthologous clusters to increase the depth of our search.
- Research: We realize that a list of putative binding sites and their possible transcription factors is not enough to diversify the fungal catalog to the point where new antibiotics and environmental toxins can now be discovered and produced. This requires a higher understanding of filamentous fungi, and the discovery of many more regulating parts. To accomplish this long term goal, more research by synthetic biologists everywhere will have to be done.
Experimental Design Summary
- Culture Aspergillus niger in nonselective solid media.
- Once plasmids and constructs are obtained, insert the constructs into the plasmids using polymerase chain reaction (PCR).
- Generate protoplasts, transform with the Tet plasmid, and apply to a new plate.
- Generate protoplasts from this new colony, and transform with the reporter plasmid.
- Confirm the presence of both constructs in the new colony.
- Add doxycycline to the media. Wait until a change in fluorescence is observed. Measure fluorescence periodically to quantify expression.
- Confirm functionality of binding sites.
- While Apsergillus niger is a biosafety level 1 organism, it is a common contaminant of food and a pathogen of multiple plant species. Care must be taken to ensure that A. niger spores are not released into the lab. This can be prevented by working in fume hoods.
Hygromycin (information taken from www.thermofischer.com)
|Category 3 acute oral toxicity|
|Category 1 serious eye damage|
|Category 1 respiratory sensitization (i.e. lung irritation)|
- Wash hands thoroughly after handling.
- Wear protective gloves/protective clothing/eye protection/face protection.
- Avoid breathing dust/fume/gas/mist/vapors/spray.
- Do not eat, drink or smoke when using this product.
- In case of inadequate ventilation wear respiratory protection.
- In the event of eye exposure, rinse eyes with water for several minutes.
- Category 2 Carcinogenicity
- Combustible Dust Hazard: May form combustible dust concentrations in the air (during processing)
Experimental Design Procedure
- (a more thorough procedure with exact measurements and reagents exists in the procedural handbook)
- We begin by culturing spores of A. niger CBS513.88 from Westerdijk Institue on 50 ml of potato dextrose agar, in a tissue plate. Then, we incubate at 30 to 37°C for 5-10 days with periodic checking, and examine it microscopically for any sign of contamination.
- After incubation, the spores will have lodged together. To dislodge them, vigorously shake your flask containing your sample and some glass beads. Once dislodged, you may remove the spores with a pasteur pipette and store them at 4°C.
Designing Genetic Constructs
- We design genetic constructs containing our putative binding sites using Benchling. These constructs are a composite of multiple genes from multiple protocols in the literature. For the sake of simplicity, there are two constructs: the tet-construct, and the reporter-construct. The tet-construct is inserted into plasmid p502, while the reporter-construct is inserted into pCT74. Both of these plasmids are then consecutively transformed into our spores.
- Our transformation procedure was adapted from a procedure by Leynaud-Kieffer et. al .
- Firstly, the cell walls of the spores are removed, and the now protoplastic spores are added to a tube along with one of the plasmids (we perform this procedure one plasmid at a time, because the rate of recombination for a plasmid in A. niger is already very low). Then, after a few short incubations, the cells are placed onto selective media for 1-2 weeks. This can then be done again for the second plasmid.
Confirmation of Functionality
- Once the transformations have successfully taken place, we treat our cells with doxycycline (a stable derivative of tetracycline), to induce the tet-on system from the tet-construct. This should, if our binding sites are in fact functional, produce a change in expression of sGFP relative to the amount of doxycycline added. To confirm that there is a change in expression, we compare fluorescence to a control sample that has been transformed in the same way. This will tell us whether our change in expression is due to doxycycline addition, or simply the addition of a new sequence to the toxA promoter.
- In order to quantify our expression, we measure sGFP on a standard curve using a protocol from BioVision .
- We did our best to predict potential setbacks for our experimental protocol, and assess them as we would if we were able to perform lab work. Below is a table of these potential outcomes:
|Possible observed outcome||Hypothesis for the mechanism behind this outcome||Hypothesis for how to overcome this challenge|
|No expression of sgfp even before the addition of doxycycline.||It has previously been observed that the ToxA promoter has been used to induce constitutive gene expression in Aspergillus niger . Constitutive expression of gfp under the ToxA promoter has been previously achieved in related species of filamentous fungi  and heterologous gfp has been recorded to be constitutively expressed in A. niger, albeit under the glaA promoter . This indicates that the ToxA-gfp expression cassette would be expected to result in sgfp expression under standard lab conditions. In the case of a lack of expression in the proposed experiment, this is likely the result of the inserted putative binding site (PBS) in the ToxA promoter, rendering the promoter nonfunctional even in the absence of the transcription factor of interest.||To support or deny this hypothesis, cells should be engineered without the proposed binding site in the ToxA promoter, though the Tet-system plasmid should still be inserted inside the cells for the sake of minimizing variables.|
|No change in expression of sgfp even after the addition of doxycycline.||The first hypothesis is that the predicted binding site simply wasn't accurate. This would indicate that our bioinformatics pipeline was insufficient for identifying filamentous fungal binding sites. The second hypothesis is that, even with the binding of the transcription factor of interest (TFOI) to the putative binding site, that the presence of the transcription factor did not sufficiently impede the native ToxA transcriptional machinery enough to result in a change in sgfp expression. This could be because the TFOI binding site was too far away from the ToxA TATA box, or perhaps that the binding of the ToxA-associated transcription factors to ToxA impeded the TFOI's ability to bind to the PBS.||In the first scenario, the next steps would be to review the score given to the binding site by our software. We may need to increase our scoring threshold,
or re-run our software with better parameters. This may involve gathering more orthologous clusters or shortening the promoter lengths, in order to increase the
depth and specificity of the MEME search.
In the case of the TFOI simply not sufficiently altering sgfp transcription, testing of this hypothesis would involve placing the PBS on various parts of the ToxA promoter and seeing which PBS locations result in a change in sgfp expression.
|No viable colonies were observed on the selective media||The strength of the gpdA promoter in Aspergillus niger, which controls hygromycin resistance in our plasmid pCT74, is well documented, and is considered sufficient to select for viable colonies in a transformation experiment . While Pcox4 was demonstrated to have lower levels of expression than PgpdA, it is nevertheless within that same range of expression and should be sufficient to confer antibiotic resistance on transformed cells . This would refute the hypothesis that promoter strength for our selectable markers was inadequate to preserve cell viability. If no viable colonies were observed, the likeliest hypothesis is that our transformation procedure had extremely low efficiency. The transformation procedure for plasmid insertion into A. niger had been previously documented by Leynaud-Kieffer et. al. , so the likeliest explanation for an unsuccessful transformation would be human error on our part.||Review the transformation procedure obtained from Leynaud-Kieffer et. al. and adapted for our purposes, and check to see if any changes made would have had detrimental effects on the transformation efficiency. Perform the transformation again, with more experienced lab members and under closer supervision, and observe whether an increased number of viable colonies is observed afterwards.|
|sGFP takes too long to degrade||sGFP is highly stable with a half life of 18 hours which means it may take quite a long time for us to see any changes in fluorescence when transcription is blocked by transcription factor binding. This could hinder the overall timeliness and accuracy of the experiment. ||In order to decrease the time it takes sGFP to degrade, we would need to find a species specific proteolytic degradation targeting sequence which we would put onto our sGFP gene. This way the tag would recruit proteases which would shorten the half life of sGFP.|
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