Team:BioIQS-Barcelona/DryLab
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Dry Lab | Overview
Have a look!Inner beauty
Despite what some people may think, DryLab occasionally may be equal or more important for a project to develop properly than the WetLab. Applying this thought to our project, we decided to study some important aspects and evaluate their viability. More specifically, our work in the DryLab consisted on:
1st Modelling
We had several ideas about how the sensor could work, and eventually, one rose up strongly. The first step when you have an idea, goes through think about it, understand it and rethink it as many times as it is required. Maybe seems an intuitive reflection, but constitutes the fundamental part of every development. Using mathematical models and computer simulations we have been able to understand the dynamics underlying our sensor, variety of parameters could be tuned, eventualities could be tested and tendencies could be predicted.
Generally speaking, the bottleneck of our idea or in other words, the critical working point, was to be able to build up a complex gold nano-particles network. In order to enable an easy optical detection. Understanding the implications associated, makes the difference when, at the end, real design must be faced. Keep reading if you want to know our sensor. How HLA-DQ and gluten-derived peptides interactions could be detected? how this detection could be easy and visual? And finally, which is the physical dynamics pervading?
2nd PCRs standardization
One of the strengths of our approach to detect patient-specific reactive gluten peptides in food is that it will only be necessary to obtain a DNA sample (saliva) from the patient. Patient’s HLA-DQ (the protein responsible of recognising and triggering the immune response) will be expressed using a PCR protocol developed in the WetLab. For this purpose, we use specific primers to amplify patient’s target genes and thus, it is necessary to evaluate if they are useful for all of the different celiac-associated genotypes.
A multiple sequence alignment (MSA) on all annotated celiac-associated genomic sequence was performed to evaluate the specific primer-binding regions conservation.
It has been demonstrated that these regions are perfectly conserved or slightly different for both DQ2 and DQ8 celiac haplotypes separately. This means that either the same primer can be used for amplifying all genotypes or that a specific DQ2- or DQ8-primer has to be used.
3rd Molecular recognition
Another aspect that we wanted to evaluate is how the HLA-DQ protein is able to effectively recognise and bind gluten-derived peptides. Specifically, which are the residues that are mediating this recognition and, if they are conserved among celiac haplotypes DQ2 and DQ8.
To do so, we focused the conservation study to those residues that have been suggested to be responsible of the peptide recognition. A multiple alignment on protein sequences was performed using all the annotated so far.
Three different residues, two in the α chain and one in the β chain, have been determined as conserved among celiac patients and absent in healthy people and hence, it has been established that this mutated residues strictly need to be present in the HLA protein in order to molecularly recognise a gluten peptide and develop the disease.