Team:BioIQS-Barcelona/DryLab
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 to our project, we decided to study some important aspects and evaluate their viability. More specifically, our work in the DryLab consisted on:
1st: Modeling
We had several ideas about how the sensor could work, and eventually, one rose up strongly. The first step is having an idea. It may seem simple, but it can take long, as it goes through think about the idea, understand it and rethink it as many times as it is required. Maybe this reflection seems intuitive, but constitutes the fundamental part of all developments. Using mathematical models and computer simulations we have been able to understand the dynamics underlying our sensor. Furthermore, using this model we can tune a huge variety of parameters, test many eventualities and predict the tendency of the sensor.
Generally speaking, the bottleneck or critical working point of our idea, was being able to build up a complex network of gold nano-particles. This would enable our sensor to have an easy optical detection system. Understanding the implications associated in that procedure makes the difference while facing the real design. How can we detect the interactions between the HLA-DQ protein and gluten-derived peptides? How can this detection be easy and visual? And finally, which are the physical dynamics pervading in the system? Go to the modeling section if you wish to know the answer to all this questions.
2nd: PCRs standardization
One of the strengths of our approach to detect patient-specific reactive gluten peptides in food is that we can produce the sensor using only a DNA sample from the saliva of the patient, which is not invasive. Patient’s HLA-DQ (the protein responsible for recognizing and triggering the immune response) is then expressed using the PCR protocol designed in the WetLab. For this purpose, we use specific primers to amplify the patient’s target genes and thus, it is necessary to evaluate if they will be 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 conservation between genotypes of the specific primer-binding regions.
The results demonstrate that this regions are perfectly conserved or only slightly different for both DQ2 and DQ8 celiac haplotypes. 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. The possibility to use the same primers for multiple patients reduces the costs associated in the personalization factor. Thus, we've created a standard method to achieve the required personalization.
3rd: Molecular recognition
Another aspect that we wanted to evaluate is how the HLA-DQ protein is able to effectively recognize 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 the residues that have been previously suggested to be responsible for the peptide recognition. A multiple alignment of protein amino acid sequences was performed using all the annotated HLA-DQ proteins so far.
The results demonstrate that three different residues, two in the α chain and one in the β chain, are conserved among celiac patients and absent in healthy people. This mutated residues strictly need to be present in the HLA-DQ protein in order to molecularly recognize a gluten peptide and develop the disease. Therefore, we elucidated which are the residues of the HLA-DQ protein responsible for its interaction with the epitopes involved in celiac disease.