Team:Heidelberg/Micrococcal Nuclease


Micrococcal Nuclease





DNA degradation Assay

Background

In the discussion with Dr. Kölker we expanded our idea of the treatment by natural transformation for the gut microbiome to the lung microbiome. Over 30.000 people suffer worldwide from the life-limiting, recessive disease called cystic fibrosis (CF) and approximately 1.000 get such a diagnosis annually [1].
These high numbers of cases are reason enough to have a closer look on the cystic fibrosis disease. Mutations cause a defection in the cystic fibrosis transmembrane conductance regulator (CFTR) protein which is an anion channel in the apical membrane of respiratory, digestive, reproductive and sweat glands epithelium. The CFTR protein usually lets chloride and bicarbonate pass the apical membrane, regulates water and ion transport and maintains epithelial surface hydration. Furthermore, the release of bicarbonates in the airway is essential for the proper unfolding of mucins and defending against bacteria. Thus, a defection in the CFTR protein leads to mucosal hyperconcentration in the respiratory, digestive and reproductive systems and malabsorption of chloride and sodium in the sweat glands [2]. If CF does not get treated properly e.g. a severe bronchiectasis will break out and lead to symptoms like chronic coughs, coughing up blood, chest pain, male infertility, salt loss syndromes and many more [3]. Moreover, most people with CF become symptomatic at birth or soon after birth.
So far researchers have come far from viewing the diseases as deathly to enabling many young adults a productive life but there are still patients with rare CFTR mutations that can be helped that easily [4]. One commonly used medication against CF is Dornase alfa (proprietary name Pulmozyme®). Dornase alfa is a highly purified solution of recombinant human deoxyribonuclease 1 (rhDNase) which causes a reduction of the mucus viscosity in the lungs and promotes an improved clearance of secretion. In our approach we want to use a Micrococcal Nuclease, which is a DNA and RNA endonuclease with the ability to degrade double-stranded and single-stranded DNA and RNA. The micrococcal nuclease is ideal for degradation of nucleic acid in crude cell-free extracts and therefore perfectly suitable for our approach [5]. Due to time limitation we ordered the plasmid from Addgene GST-MNase (Plasmid #136291) with the vector backbone pGEX-6P-1 [6].

Figure 1: Schematic description of micrococcal nuclease (MNase) assay. The design of the MNase assay was to control the degraded DNA in the gel. We cultured Escherichia coli with MNase and for the negative control with mCherry overnight and took samples directly from the medium at time point 0 and after about 12 hours. Created with Biorender.com

Experimental results

We performed the assay to detect the function of the MNase twice but the results were equal. In figure 2 our results are demonstrated. In our positive culture 2 with the MNase we observed a loss of the DNA band at about 2 kb but the negative control with the E. coli expressing the mCherry fluorescence protein also showed a degradation of the plasmid.

Figure 2: Micrococcal nuclease (MNase) gel results. In the gel the the 1 mL overnight cultures are compared one with Escherichia coli DH5ɑ with plasmid MNase (culture 2) against E. coli BL21 with mCherry (culture 1). In both cultures about 3x10^3 circular and linear dsDNA was added. 10 µl was taken at the time point 0 and 12 h later from the LB medium first without bacteria and after 12 h after centrifugation 3.000 rpm for 2 min.

Discussion

The control of the MNase function was a qualitative and fast designed assay. The results show no concrete trend for or against the function of the MNase because the negative control showed positive results. Reasons for this could be that dsDNA degrades in a solution at 37 °C for over 10 hours automatically. Or the bacteria could digest and use the DNA naturally with another DNase. Additionally, the secretion of the MNase must be controlled before in an SDS-PAGE for example to compare the band in the supernatant versus the lysate and pellet.
To summarize this chapter with the MNase is that our assay must be improved in order to prove the MNAse presence in the reaction solution, its function and finally also its efficiency. This chapter was made to reach further diseases to tackle with our application. Therefore, it is necessary to test the efficiency of MNAse to degrade not only dsDNA but show that MNase efficiency to liquefy Mucus from cystic fibrosis patients in a comparable efficiency as the existing drug Pulmozyme®.

References

[1] About Cystic Fibrosis, https://www.cff.org/ (retrieved on 19.10.2021)

[2] Radlović N. (2012). Cystic fibrosis. Srpski arhiv za celokupno lekarstvo, 140(3-4), 244–249.

[3] De Boeck, K., Vermeulen, F., & Dupont, L. (2017). The diagnosis of cystic fibrosis. Presse medicale (Paris, France : 1983), 46(6 Pt 2), e97–e108. https://doi.org/10.1016/j.lpm.2017.04.010

[4] De Boeck K. (2020). Cystic fibrosis in the year 2020: A disease with a new face. Acta paediatrica (Oslo, Norway : 1992), 109(5), 893–899. https://doi.org/10.1111/apa.15155

[5] Properties of Exonucleases and Non-specific Endonucleases, https://www.nebiolabs.com.au/tools-and-resources/selection-charts/properties-of-exonucleases-and-nonspecific-endonucleases2 (retrieved on 19.10.2021)

[6] Mapping Native R-Loops Genome-wide Using a Targeted Nuclease Approach. Yan Q, Shields EJ, Bonasio R, Sarma K. Cell Rep. 2019 Oct 29;29(5):1369-1380.e5. doi: 10.1016/j.celrep.2019.09.052. 10.1016/j.celrep.2019.09.052 PubMed 31665646