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Update Group C #54

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Oct 28, 2023
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Update Group C #54

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ecc74d8
Added graphics and updated writeup except for table
daniel-panhead Oct 25, 2023
d3ddf72
Added table with data
daniel-panhead Oct 25, 2023
6a0928f
Updated results, discussion, and references
daniel-panhead Oct 26, 2023
c7f1967
Prettified Code!
daniel-panhead Oct 26, 2023
747dbb5
add changes to the discussion content
IcedTea2K Oct 27, 2023
c44f965
Prettified Code!
IcedTea2K Oct 27, 2023
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Made graph bigger
daniel-panhead Oct 28, 2023
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2 changes: 1 addition & 1 deletion src/components/Equation.vue
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Expand Up @@ -12,7 +12,7 @@ onMounted(() => {
</script>
<template>
<div
class="w-full overflow-x-scroll whitespace-nowrap bg-transparent invisible-scroller text-center"
class="w-full overflow-x-scroll bg-transparent invisible-scroller text-center"
ref="containerRef"
></div>
</template>
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223 changes: 199 additions & 24 deletions src/views/lab-validation/GroupCView.vue
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Expand Up @@ -59,21 +59,34 @@ const sectionTitleStyle = 'text-subtitle-sm lg:text-subtitle text-white mb-4'
thiolation and prepare it for future reactions.
</p>
<CaptionedGraphics modifier="w-full">
<template #graphics>
<img src="../../assets/thiolation-and-conjunction/reaction-of-sata.png" alt="" />
</template>
<template #caption>
<p>Figure 1: Reacting SATA with amine to generate sulfhydryl group.</p>
<p>
Figure 1: Reacting SATA with amine to generate sulfhydryl group (Theule, n.d.).
</p>
</template>
</CaptionedGraphics>

<p>
Another mechanism used to conjugate the thiolated enzyme, DNase 1 to the liposome,
maleimide is the thiol-Michael addition reaction. Figure 2 depicts the sulfur group
donating its electron to the olefin of a maleimide molecule, forming a covalent bond
between the two. A proton is removed in this process, generating a thiosuccinimide
molecule that is stable under non-aqueous conditions (Quanta BioDesign, n.d.). The
Michael addition reaction allows for simple and fast modifications of cysteine
residues to conjugate specific biomolecules (Quanta BioDesign, n.d.).
The other side of the equation to conjugate the thiolated enzyme, DNase 1 to the
liposome, maleimide is the thiol-Michael addition reaction. Figure 2 depicts the
sulfur group donating its electron to the olefin of a maleimide molecule, forming a
covalent bond between the two. A proton is removed in this process, generating a
thiosuccinimide molecule that is stable under non-aqueous conditions (Quanta
BioDesign, n.d.). The Michael addition reaction allows for simple and fast
modifications of cysteine residues to conjugate specific biomolecules (Quanta
BioDesign, n.d.).
</p>
<CaptionedGraphics modifier="w-full">
<template #graphics>
<img
src="../../assets/thiolation-and-conjunction/michael-addition.png"
alt=""
class="lg:w-2/3"
/>
</template>
<template #caption>
<p>
Figure 2: Michael Addition Reaction – A thiol-maleimide reaction that forms a
Expand Down Expand Up @@ -106,11 +119,15 @@ const sectionTitleStyle = 'text-subtitle-sm lg:text-subtitle text-white mb-4'
<h3 :class="sectionTitleStyle">Preparation of HEPES Buffer Stock Solution</h3>
<p>
Start by combining 10 mM Hepes buffer (pH = 7.5), 55mM CaCl<sub>2</sub>, 10mM
MgCl<sub>2</sub>, and 0.01mM TCEP to create 100 mL of stock solution. Prepare
another batch of 1L Hepes buffer using 13.7 mM NaCl, 10 mM HEPES buffer, and 0.01 mM
TCEP at pH 6.0. This is done using 800 mL of distilled water, 2.68 g potassium HEPES
tribasic monohydrate, 523.20 mg citric acid, 0.80 g NaCl, 2.50 mg TCEP. Add 1mM HCL
to increase the pH to 6.0 and add distilled water to create 1L solution.
MgCl<sub>2</sub>, and 0.01mM TCEP to create 100 mL of stock solution.
</p>
<br />
<p>
Prepare another batch of 1L Hepes buffer using 13.7 mM NaCl, 10 mM HEPES buffer, and
0.01 mM TCEP at pH 6.0. This is done using 800 mL of distilled water, 2.68 g
potassium HEPES tribasic monohydrate, 523.20 mg citric acid, 0.80 g NaCl, 2.50 mg
TCEP. Add 1mM HCL to increase the pH to 6.0 and add distilled water to create 1L
solution.
</p>
</section>

Expand All @@ -120,8 +137,10 @@ const sectionTitleStyle = 'text-subtitle-sm lg:text-subtitle text-white mb-4'
<p>
Set up an incubator to 37°C. Combine 100 mM Tris-HCl (pH = 7.5 at 2.5°C), 25 mM
MgCl<sub>2</sub>, and 1 mM CaCl<sub>2</sub> to create a 10x Reaction Buffer with
MgCl<sub>2</sub> to create Manu Buffer. <br />

MgCl<sub>2</sub> to create Manu Buffer.
</p>
<br />
<p>
Prepare 9 vials of bovine serum albumin (BSA) and dilute it according to Table 1.
To prepare BCA working reagent (WR), the following formula is used to determine
the total volume required:
Expand Down Expand Up @@ -202,12 +221,13 @@ const sectionTitleStyle = 'text-subtitle-sm lg:text-subtitle text-white mb-4'
the thiolated DNase 1 and incubated for one hour at room temperature. This protein
solution was then diluted to 12.5 µM with a buffer consisting of 13.6 mM HCl/10 mM
citrate buffer with 0.01 mM TCEP at a pH of 6.0. This enzyme-buffer mixture was
added to freshly prepared liposomes (10 mM lipid) at a maleimide with a ratio of 0:1
to 10:1. After gentle mixing at room temperature overnight, the enzymosomes are to
be separated from unconjugated enzyme through dilution and ultracentrifuge at 176
000 x g for 1.5h at 4°C on a L8-60 M ultracentrifuge. After the supernatant is
removed, dissolve in 2 mL of citrate buffer. Store the conjugated enzymosomes at 4°C
for future usage.
added to freshly prepared maleimide-liposomes (10 mM lipid) at
liposome-to-thiolated-enzyme ratios of 1:1 and 10:1. After gentle mixing at room
temperature overnight, the enzymosomes were measured using dynamic light scattering
(DLS). Normally they would be separated from unconjugated enzymes through dilution
and ultracentrifuged at 176 000 x g for 1.5h at 4°C on a L8-60 M ultracentrifuge.
However, due to resource constraints, and the lack of an ultracentrifuge,
unconjugated enzymes were not able to be separated in this experiment.
</p>
</section>
</div>
Expand All @@ -217,7 +237,134 @@ const sectionTitleStyle = 'text-subtitle-sm lg:text-subtitle text-white mb-4'
<SingleColumn>
<template #title> Results </template>
<template #body>
The thiolated enzymes were created and were stored at 4°C for future usage.
<div class="flex flex-col gap-y-4">
<CaptionedGraphics modifier="w-full">
<template #graphics>
<img
src="../../assets/thiolation-and-conjunction/thiolated-dnase-i.png"
alt=""
class="xl:w-1/2"
/>
</template>
<template #caption>
<p>
Figure 3. Picogreen Assay Results of Thiolated DNase I. Generated DNA
concentration shown over time at 3 different ratios of SATA to DNase I (1:1, 4:1,
8:1).
</p>
</template>
</CaptionedGraphics>
<p>
For determining the most optimal ratio of the SATA to DNase 1 in order to generate the
most thiolated-DNase 1 while maintaining activity, different concentrations of SATA
were used to titrate with the targeted enzyme. According to Figure 3, thiolated DNase
1 still retain their activity. SATA: DNase 1, 1:1 and 4:1, had the greatest activity
of degrading ~250ng/mL of DNA in ~30s. 8:1 had less activity as it only degraded
~130ng/mL of DNA in the same time and never got to 0ng/mL which could suggest loss of
activity over time. 1:1 SATA: DNase 1 can be used for future thiolation procedures.
</p>
<CaptionedGraphics modifier="w-full">
<template #graphics>
<table class="table-fixed border-collapse w-full">
<tr class="bg-slate">
<th class="py-2 px-4 text-sm lg:text-base w-1/3"></th>
<th class="py-2 px-4 text-sm lg:text-base w-2/3" colspan="2">Sample Type</th>
</tr>
<tbody class="bg-dark">
<tr>
<th class="text-sm lg:text-base bg-slate"></th>
<td class="py-1 lg:text-lg text-center">
<b>1:1</b>
</td>
<td class="py-1 lg:text-lg text-center">
<b>10:1</b>
</td>
</tr>
<tr>
<th class="text-sm lg:text-base">Z-Average (d.nm)</th>
<td class="py-1 lg:text-lg text-center">74.09</td>
<td class="py-1 lg:text-lg text-center">76.77</td>
</tr>
<tr>
<th class="text-sm lg:text-base">PDI</th>
<td class="py-1 lg:text-lg text-center">0.239</td>
<td class="py-1 lg:text-lg text-center">0.254</td>
</tr>
</tbody>
</table>
</template>
<template #caption>
<p>
Table 2. Particle Size Distribution of Enzymosome Formulation. Sample types
represent formulations made at 2 ratios of 10 mM maleimide-liposomes to thiolated
DNase I (1:1 and 10:1). Samples measured using dynamic light scattering (DLS).
Z-Average (mean particle size) and PDI (polydispersity index) characterize
particle size and distribution, respectively.
</p>
</template>
</CaptionedGraphics>
<p>
The particle size of the enzymosome formulation was evaluated with a Zetasizer Nano ZS
machine to assess if the proposed conjugation of thiolated enzyme to liposomes caused
a change in particle size. As seen in Table 2, formulations containing a 1:1 ratio of
liposome to thiolated enzyme were on average about 74.09 d.nm in size, as represented
by the z-average of the samples based on intensity, and had a poly-dispersive index
(PDI) of 0.239. Similarly, formulations containing a 10:1 ratio of liposome to
thiolated enzyme were on average about 76.77 d.nm in size and had a PDI of 0.254. It
should be noted that, despite the formulation not undergoing centrifugation to
separate unconjugated enzymes, only one population of particles was observed in DLS.
</p>
</div>
</template>
</SingleColumn>

<SingleColumn>
<template #title> Discussion </template>
<template #body>
<div class="flex flex-col gap-y-4">
<p>
Conjugation of maleimide-liposomes to thiolated DNase I is inconclusive; however,
thiolation of DNase 1 was successful. It supported and confirmed the thiolation
modelling’s findings that thiolation will not occur in the active site. Nevertheless,
there was some reduction in activity at 8:1, SATA: DNase I ratio. A possible reason
for the lower activity of the 8:1 could be due to the excess thiolation of the enzyme
which causes deformation within the 3D structure. Changes in the structure and
polarity of side groups could lead to changes elsewhere in the protein. For instance,
those changes could occur at the active site, which explains why the optimation to
ensure DNase 1 is thiolated but not in excess is critical. Different enzymes and
proteins may have optimal SATA ratios to ensure through thiolation.
</p>
<p>
According to Table 2, in both maleimide-liposomes to thiolated DNase I 1:1 and 10:1
ratios, the mean particle size was 74.09 d.nm and 76.77 d.nm respectively and the
Z-Average increased notably to the mean particle size of the maleimide-liposomes only,
32.36 d.nm. The increase in the mean particle size suggests the conjugation of the
thiolated DNase I to maleimide-liposomes. Additionally, the PDI of both 1:1 and 10:1
ratios, 0.239 and 0.254 respectively, were higher than the PDI of only
maleimide-liposomes, 0.206 (Table 2 & Liposome Formation, Table 1). As PDI refers to
the distribution of molecular weights in a given solution, the higher PDI indicates
the presence of multiple particle sizes. In other words, in this case refers to the
presence of thiolated DNase I, liposomes, and conjugated maleimide-liposomes to
thiolated DNase I in the given solution. There was not a substantial difference
between the mean particle sizes of 1:1 and 10:1 ratios and despite that the given
solution had not undergone purification, only one population was observed in DLS. The
regularization method in DLS presumes the presence of multiple particle populations
and could detect the particles if their sizes are 3-5 times different within the
detection limits of the DLS (Wyatt Technology, n.d.). This technique can detect
particle sizes approximately between 0.3nm-10µm (Raval et al., 2019). By using the
molecular weight of DNase I (30.33 kDa) and the partial specific volume (0.73 cm³/g),
the approximate diameter of DNase I is calculated to be 4.13 nm (Parsiegla et al.,
2013 & Erickson, 2009). The reason why only one population of particles was observed
in DLS could be that the subpopulations did not scatter the light strongly and only
resulted in one peak (Filipe et al., 2010).
</p>
<p>
To further verify the conjugation of the maleimide-liposomes to thiolated DNase I,
techniques such as cyro-TEM (transmission electron microscopy), cyro-SEM (scanning
electron microscopy, and size exclusion chromatography could be used before making
conclusions regarding its effectiveness.
</p>
</div>
</template>
</SingleColumn>

Expand All @@ -229,6 +376,17 @@ const sectionTitleStyle = 'text-subtitle-sm lg:text-subtitle text-white mb-4'
Deng, Y., & Tsao, B. P. (2013). Genetics of Human SLE. Dubois’ Lupus Erythematosus and
Related Syndromes, 35–45. https://doi.org/10.1016/b978-1-4377-1893-5.00004-2
</p>
<p>
Erickson, H. P. (2009, May 15). Size and shape of protein molecules at the nanometer
level determined by sedimentation, gel filtration, and electron microscopy. Biological
procedures online. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3055910/
</p>
<p>
Filipe, V., Hawe, A., & Jiskoot, W. (2010, May). Critical evaluation of nanoparticle
tracking analysis (NTA) by NanoSight for the measurement of nanoparticles and protein
aggregates. Pharmaceutical research.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2852530/
</p>
<p>
Instructions SATA and SATP. Thermo Scientific. (n.d.).
https://assets.fishersci.com/TFS-Assets/LSG/manuals/MAN0011179_SATA_SATP_UG.pdf
Expand All @@ -239,18 +397,35 @@ const sectionTitleStyle = 'text-subtitle-sm lg:text-subtitle text-white mb-4'
liposomes. BBA-General Subjects, 1117(3), 258-264. Doi: 10.1016/0304-4165(92)90022-M
</p>
<p>
Kishi, K., Toshihiro, Y., Takeshita, H. (2001) DNase I: structure, function, and use
in medicine and forensic science. Legal Medicine, 3(2), 69-83. doi:
Kishi, K., Toshihiro, Y., & Takeshita, H. (2001). DNase I: structure, function, and
use in medicine and forensic science. Legal Medicine, 3(2), 69-83. doi:
10.1016/S1344-6223(01)00004-9
</p>
<p>
Maleimide Reaction Chemistry. Quanta BioDesign. (n.d.).
https://www.quantabiodesign.com/maleimide-reaction-chemistry/
</p>
<p>
Parsiegla, G., Noguere, C., Santell, L., Lazarus, R. A., & Bourne, Y. (2013, January
9). 4AWN: Structure of recombinant human dnase I (rhdnasei) in complex with magnesium
and phosphate. RCSB PDB. https://www.rcsb.org/structure/4awn
</p>
<p>
Raval , N., Maheshwari, R., Kalyane, D., Youngren-Ortiz , S. R., Chougule, M. B.,
Tekade, R. K., 6, & AbstractWith increasing importance of nanoparticles in
pharmaceutical applications. (2019, January 11). Importance of physicochemical
characterization of nanoparticles in pharmaceutical product development. Basic
Fundamentals of Drug Delivery.
https://www.sciencedirect.com/science/article/pii/B9780128179093000108
</p>
<p>
Theule, Stephanie. (n.d.). Reaction of SATA [Reaction Image]. ResearchGate.
https://www.researchgate.net/figure/SATA-and-its-reaction-with-primary-amines-SATA-reacts-with-primary-amines-and_fig18_29529224
</p>
<p>
Understanding Dynamic Light Scattering. Waters | Wyatt Technology. (n.d.).
https://www.wyatt.com/library/theory/dynamic-light-scattering-theory.html#:~:text=In%20dynamic%20light%20scattering%20(DLS,to%20the%20particles%27%20hydrodynamic%20radii
</p>
</div>
</template>
</SingleColumn>
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