Molecular research into the use of mycelial compounds


#1

Hello,

My name is Berend and currently doing my thesis in Biomolecular Sciences on the topic of mycelial compounds. Since my field is specialised in molecular biology, I will focus mainly on the molecular aspects of this interesting topic.

The current goal is to observe the effects of citrus additives on gene expression levels of wood degrading enzymes (such as Laccases, Lignin peroxidases & Manganese peroxidases) and to follow growth (using a calorimeter and/or observing growth rate by looking at the diameter on a petri dish) on substrate to will eventually be used in end products.

How it all started

I am the successor of someone who, for his research, laid the foundation on which I am building now. One of his findings was that the addition of mandarin peelings (or peeling extract by autoclaving water with mandarin peelings), induced the production of 2 paralogues of laccases (Lac4 & 5). These findings were mainly supported based on qRT-PCR results.

My own research

The thesis of my predecessor would prove to be the beginning of my own research as I wanted to further these findings by looking at more paralogues of the laccases as well as looking at other substrate degrading enzymes. To this extent, I started by designing primers based on the available genome for the organism on which I will be working: Trametes versicolor (https://www.ncbi.nlm.nih.gov/genome/3260).
Primer pairs were generated for all paralogues of the laccase (Lac) family (except 4 & 5), lignin peroxidases (LiP) & manganese peroxidase (MnP) using the practical NCBI primer blast tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/). These primers needed to be used for qRT-PCR, so the product was sought to be between 100-200bp long. The primers itself were generated to be around 20bp and specific to the target sequence, with a GC content between 40-60%, a melting temperature of optimally 60°C and they should contain a G or C at the 3’ and 5’ end as to promote binding and hence make it more efficient.
To see if the sequences would make secondary structures, IDT’s oligo analyser tool was used (https://eu.idtdna.com/calc/analyzer).
5 pairs for each family were ordered and screened on regular DNA (extracted via the fast microwave method as laid out by my predecessor). The following picture depicts the findings of a preliminary screening using regular PCR conditions of the Kappa-HiFi protocol for 2 strains of Trametes versicolor (M99 & SV1):


SV1

M99

What I am looking for here is a single DNA band around the expected size (100-200bp), so that is to say, between the first and second band of the molecular weight marker.
The findings:
SV1: Lac6, LiP3, LiP5 & MnP3 seem promising. Annealing temperature needs to be adjusted for Lac6, LiP5 & MnP3.
M99: Lac1, Lac6, LiP3, MnP3 & MnP5 seem promising. Temperature adjustments needed for LiP3, MnP5.

A temperature gradient (between 67 & 75°C) PCR was performed on the aforementioned primer pairs. Results are depicted below:


Temperature adjustments were necessary as can be seen by the appearance of single bands all of a sudden.

With the primer pairs adjusted to the right temperature this gives the following list of genes to be researched:
M99:
~Lac1 @ 65
Lac6 @ 65
LiP3 @ 73-74
MnP5 @ 73-74

SV1:
Lac6 @ 70
LiP3 @ 65
LiP5 @ 67
~MnP1 @ 65
MnP3 @ 67-74

What comes next

  • Primer efficiency needs to be looked into before continuing to qRT-PCR. Hereby different primer pair dilutions will be used and plotted against obtained product.

  • Growing of strains on substrates with/without citrus additives to be used for calorimeter.

Preliminary testings for growing the strains on substrates to which additives are added are running. Growth is being monitored on multiple media. So far on the medium: Agar in which hemp are added, which were made by using peeling extracts as liquid, showed promising results: the plates with the peeling extracts showed a remarkable increase in growth speed compared to the control. Currently growth is also monitored on hemp medium alone (supplemented with 0.1-1% dried fruit peelings). Difference is minimal thus far (2 days after inoculation). Results will follow soon.

This is, at the moment, the main scope of my findings till now.

Thanks!

Creative Commons License
Master Thesis by Berend Basseleer, Under supervision of Elise Elsacker, Prof. Lars De Laet and Prof. Eveline Peeters / Vrije Universiteit Brussel / Department of Architectural Engineering and Bioengineering Sciences. This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License.


#2

Hi @BerendBassleer, welcome and thanks for sharing this impressive first post with us!

As a bioengineer, I hope I get what you’re trying to do here: correlating a specific location (or sequence of DNA) in the genetic code of the fungus with an increased activity of the enzymes you’re interested in. Am I close?

Would you be willing to explain exactly what you’re doing in layman terms for the many people non-schooled in biotech? :slight_smile: