- Prior to using any enzymes in creating an enzymosome, the baseline activity of each - enzyme in different solvent contexts must be determined to control for the effects of - various solvents on enzyme activity. -
--
- The enzyme DNase I has been selected for enzymosome creation, as it plays a key role in - degrading biofilms. DNase I disrupts bacterial biofilms by degrading the extracellular - DNA within the biofilm, which can be used as a nutrient source by the bacteria that - produce the biofilm (Nijland et al., 2010). -
- - - -Figure 1. blah blah blah
++ Prior to using any enzymes in creating an enzymosome, the baseline activity of each + enzyme in different solvent contexts must be determined to control for the effects of + various solvents on enzyme activity. This is important for determining whether they + will interfere with enzyme function, as well as which solvent is best for working with + the enzyme. +
++ The enzyme DNase I has been selected for enzymosome creation, as it plays a key role + in degrading biofilms. DNase I disrupts bacterial biofilms by degrading the + extracellular DNA within the biofilm, which can be used as a nutrient source by the + bacteria that produce the biofilm (Nijland et al., 2010). +
+DNA I Assay
-- The DNase I assay was adapted from the standard protocol of the Picogreen Assay kit - (ThermoFischer Scientific, 2022). This kit uses a reaction buffer containing 1 M - Tris-HCl (pH 7.5), 1 M MgCl2, and 1 M CaCl2 (ThermoFischer Scientific, 2022), which will - be referred to as the manufacturer’s buffer. 0.05 U/mL and 0.1 U/mL DNase I solutions - were prepared in the manufacturer’s buffer and the HEPES buffer separately for use in - the activity assay. 0.05 U/mL and 0.1 U/mL concentrations of DNase I in HEPES buffer - contained 55mM CaCl₂ and 10mM MgCl. The HEPES buffer was also used for thiolation - treatment and conjugation to the liposome. -
--
- 1 μM DNA Probe was added to six wells of the 96 well plate to generate 0, 4, 8, 12, 16, - and 20 pmol/well standards of 50 μL total volume. The manufacturer’s buffer, the HEPES - buffer, and DNase I with the manufacturer’s buffer and DNase I with the HEPES buffer - were added to separate wells of the 96-well plate, with each solution being added to two - wells and 50 μL of solution in each well. These were the negative and experimental - controls. 50 μL of molecular biology grade water was also added to two wells as a - background control. 50 μL of only Sample Reaction Mix was also added to two wells as a - positive control. 50 μL of Sample Reaction Mix was also added to each of the negative, - background, experimental and positive control wells for a total of 100μL of solution in - each well. Fluorescence was measured at an excitation wavelength of about 480 nm and an - emission wavelength of about 520 nm to determine enzyme activity using a - fluorometer-spectrophotometer in kinetic mode every minute for at least 10 minutes. -
++ The DNase I assay was adapted from the standard protocol of the Picogreen Assay kit + (ThermoFisher Scientific, 2022). This kit uses a reaction buffer containing 1 M + Tris-HCl (pH 7.5), 1 M MgCl2, and 1 M CaCl2 (ThermoFisher + Scientific, 2022), which will be referred to as the manufacturer’s buffer. 0.05 U/mL + and 0.1 U/mL DNase I solutions were prepared in the manufacturer’s buffer, the TE + buffer and the HEPES buffer separately for use in the activity assay. Buffer DNase + concentration is given in units of DNase activity per mL (U/mL), with the activity + unit defined as the complete degradation of 1 µg of plasmid DNA at 37°C by one unit of + DNase I in 10 minutes (ThermoFisher Scientific, 2011). The HEPES buffer was also used + for thiolation treatment and conjugation to the liposome. +
++ An aqueous working solution of the Quant-iT™PicoGreen™ dsDNA Reagent was prepared in + the TE buffer by using the buffer to dilute it 200 fold. A 2 µg/mL stock solution of + lambda dsDNA was prepared in the TE buffer as well by using the buffer to dilute it 50 + fold. In a 96 well plate, the controls were created by adding water, the + manufacturer's buffer, the TE buffer, and the HEPES buffer to twelve wells such that + each solution type had three wells of 80 µL each. For the other treatments, 40 µL of + the aqueous working solution of the Quant-iT™ PicoGreen™ dsDNA Reagent was added + each well, followed by the designated amount of buffer for each treatment shown in + Table 1 as well as 4 µL of the stock solution of lambda dsDNA. The wells were + incubated for five minutes at room temperature with protection from light before + adding the corresponding volume of DNase I indicated in Table 1. Fluorescence was + measured at an excitation wavelength of about 480 nm and an emission wavelength of + about 520 nm to determine enzyme activity using a fluorometer-spectrophotometer in + kinetic mode every 2 minutes for at least 10 minutes. These treatments were performed + three times to obtain a triplicate. +
++ Table 1: Designated buffer and DNA volumes for each treatment. +
+- Figure 1: Fluorescence of the DNase I Picogreen assay in the manufacturer’s - buffer, HEPES buffer and TE buffer. -
+ +
- Figure 2: Calibration curve of the DNase I picogreen assay at DNA concentrations - of 1, 10, 25, 50 and 100 ng/mL. -
+
+ + Figure 1. Exhibits the DNase I Picogreen Assay in Manufacturer, HEPES, and TE buffers. + It displays the generated DNA concentration in testing the activity of DNase I in + different buffers. Panels a, b, and c display Manufacturer, HEPES, and TE buffers + respectively. +
- Table 1: Approximate concentration of DNase I in each buffer at the beginning and +
+ Table 2. Approximate concentration of DNase I in each buffer at the beginning and end of the fluorescence measuring period, based on the combined information of Figure 1 and Figure 2.
+ Figure 1 demonstrates the DNA concentration over time in solutions with various + concentrations of DNase I and various buffers. This concentration appears to be + relatively constant in the TE buffer, while decreasing gradually over time in the + manufacturer’s buffer and the HEPES buffer. +
Since DNase I is an enzyme that degrades DNA (Nijland et al., 2010), the concentration - of DNA determined from Figures 1 and 2 demonstrates that DNase I has minimal activity - in the TE buffer and a relatively high amount of activity in the manufacturer's buffer - and HEPES buffer. These results are supported because the EDTA in the TE buffer + of DNA determined from Figure 1 demonstrates that DNase I has minimal activity in the + TE buffer and a relatively high amount of activity in the manufacturer's buffer and + HEPES buffer. These results are supported because the EDTA in the TE buffer inactivates DNase by chelating with the metal cations it needs to function (Panda et al., 2019), causing less DNase I activity in the TE buffer than in the other buffers.
+Future Directions
+- While only DNAse I has been selected, a combination of enzymes would ideally be used + While only DNase I has been selected, a combination of enzymes would ideally be used in conjunction on the enzymosome to combat bacterial biofilms. For example, alpha amylase and alginate lyase inhibit biofilm formation by degrading the polysaccharides that compose the biofilm (Lahiri et al., 2021; Zhu & Yin, 2015), and incorporating @@ -189,40 +238,76 @@ const tableData = {
Dische, Z. (1953). Qualitative and quantitative colorimetric determination of heptoses. Journal of Biological Chemistry, 204(2), 983–997. + https://doi.org/10.1016/S0021-9258(18)66101-0
Lahiri, D., Nag, M., Banerjee, R., Mukherjee, D., Garai, S., Sarkar, T., Dey, A., Sheikh, H. I., Pathak, S. K., & Edinur, H. A. (2021). Amylases: Biofilm inducer or biofilm inhibitor? Frontiers in Cellular and Infection Microbiology, 11, 660048. + https://doi.org/10.3389/fcimb.2021.660048
Nijland, R., Hall, M. J., & Burgess, J. G. (2010). Dispersal of biofilms by secreted, matrix degrading, bacterial DNase. PloS One, 5(12), e15668. + https://doi.org/10.1371/journal.pone.0015668
Panda, B. B., Meher, A. S., & Hazra, R. K. (2019). Comparison between different methods of DNA isolation from dried blood spots for determination of malaria to determine specificity and cost effectiveness. Journal of Parasitic Diseases, 43(3), 337–342. + https://doi.org/10.1007/s12639-019-01136-0
Preiss, J., & Ashwell, G. (1962). Alginic acid metabolism in bacteria: I. Enzymatic formation of unsaturated oligosaccharides and 4-deoxy-L-erythro-5-hexoseulose uronic acid. Journal of Biological Chemistry, 237(2), 309–316. + https://doi.org/10.1016/S0021-9258(18)93920-7 +
++ Scopes, R. K. (2001). Enzyme activity and assays. Encyclopedia of Life Sciences. + https://doi.org/10.1038/npg.els.0000712
-Scopes, R. K. (2001). Enzyme activity and assays. E LS.
Sigma-Aldrich. (2014). Amylase Activity Assay Kit. - https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/377/793/mak009bul.pdf + https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/377/793/mak009bul.pdf +
++ ThermoFisher Scientific. (2011). COA: DNase I, RNase-free, #EN0521. + https://www.thermofisher.com/document-connect/document-connect.html?url=https%3A%2F%2Fassets.thermofisher.com%2FTFS-Assets%2FLSG%2Fbrochures%2Fcoa_en0521.pdf
- ThermoFischer Scientific. (2022). Quant-iTTM PicoGreenTM dsDNA Reagent and Kit. - https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets%2FLSG%2Fmanuals%2Fmp07581.pdf + ThermoFisher Scientific. (2022). Quant-iTTM PicoGreenTM dsDNA Reagent and Kit. + https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets%2FLSG%2Fmanuals%2Fmp07581.pdf
Zhu, B., & Yin, H. (2015). Alginate lyase: Review of major sources and classification, properties, structure-function analysis and applications. Bioengineered, 6(3), 125–131. + https://doi.org/10.1080/21655979.2015.1030543