Difference between revisions of "Part:BBa K5237017"
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   <!-- Part summary -->
 
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   <section id="1">
 
   <section id="1">
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     <h1>FRET-Acceptor: TetR-mScarlet-I</h1>
+
     <h1>FRET-Donor: mNeonGreen-Oct1</h1>
−
     <p>This composite part is a fusion protein of TetR and the red fluorescent protein mScarlet-I. It was used as a FRET
+
     <p>This composite part is a fusion protein of Oct1-DBD and mNeonGreen. It was used as a FRET donor in combination
−
       acceptor in combination with mNeonGreen-Oct1 as the donor.
+
       with tetR-mScarlet-I as an acceptor.
−
       This part was used to measure the proximity of two DNA strands by FRET fluorescence measurements.
+
       This part was used to measure the proximity of two DNA strands by FRET fluoresence measurements.
 
     </p>
 
     </p>
 
     <p>&nbsp;</p>
 
     <p>&nbsp;</p>
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         <ul>
 
         <ul>
 
           <li class="toclevel-2 tocsection-2.1"><a href="#2.1"><span class="tocnumber">2.1</span> <span
 
           <li class="toclevel-2 tocsection-2.1"><a href="#2.1"><span class="tocnumber">2.1</span> <span
−
                 class="toctext">TetR DNA binding protein</span></a>
+
                 class="toctext">Oct1-DBD</span></a>
 
           </li>
 
           </li>
 
           <li class="toclevel-2 tocsection-2.2"><a href="#2.2"><span class="tocnumber">2.2</span> <span
 
           <li class="toclevel-2 tocsection-2.2"><a href="#2.2"><span class="tocnumber">2.2</span> <span
−
                 class="toctext">mScarlet-I</span></a>
+
                 class="toctext">mNeonGreen</span></a>
 
           </li>
 
           </li>
 
           <li class="toclevel-2 tocsection-2.3"><a href="#2.3"><span class="tocnumber">2.3</span> <span
 
           <li class="toclevel-2 tocsection-2.3"><a href="#2.3"><span class="tocnumber">2.3</span> <span
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K5237016 SequenceAndFeatures</partinfo>
 
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   <h1>2. Usage and Biology</h1>
 
   <h1>2. Usage and Biology</h1>
 
   <section id="2.1">
 
   <section id="2.1">
−
     <h1>2.1 TetR DNA binding protein</h1>
+
     <h1>2.1 Oct1-DBD</h1>
−
     <p>
+
     <p>Oct1-DBD is the DNA-binding domain of the human transcription factor Oct1 (POU2F1), which plays a key role in
−
      The tetracycline repressor protein (tetR) is naturally present in gram-negative bacteria and is involved in the
+
      gene regulation, immune response, and stress adaptation in eukaryotic cells. This domain specifically binds to the
−
       resistance mechanism against tetracycline (and derivatives). It does so by tightly controlling the gene expression
+
       octamer motif (5'-ATGCAAAT-3') within promoter and enhancer regions, influencing transcriptional activity
−
      of <i>tetA</i>, which encodes an efflux pump responsible for removing tetracycline from the cell.
+
       (Lundbäck <i>et al.</i>, 2000). The Oct1-DBD consists of both a POU-specific domain and a POU homeodomain, which
−
       TetR binds selectively to two plaindromic recognition sequences (<i>tetO</i>>1,2) with high affinity. For DNA
+
       work
−
      binding to occur tetR adopts a homodimeric structure and binds with two &#945;-helix-turn- &#945;-helix motifs
+
       together to form a stable complex with DNA (Park <i>et al.</i>, 2013, Stepchenko <i>et al.</i> 2021).
−
       (HTH) to two tandemly oriented tetO sequences. In the presence of tetracycline or its analogs, tetR undergoes a
+
    </p>
−
       conformational change, which prevents it from binding to DNA, therby allowing gene expression(Orth <i>et al.</i>
+
    <p>In synthetic biology, Oct1-DBD was previously used for plasmid display technology due to its strong binding
−
      2000; Kisker <i>et al.</i> 1995).
+
       affinity (K<sub>D</sub> = 9 &#215; 10<sup>-11</sup> M). Proteins fused with Oct1-DBD showed increased expression
−
      <br>
+
       and protein solubility
−
      Due to its robust and highly regulatable DNA-binding properties, tetR has become a widely adopted tool in
+
       (Parker <i>et al.</i> 2020).
−
       synthetic
+
−
       biology. Its ease of modification and ability to function in both prokaryotic and eukaryotic systems have made it
+
−
       an essential element in the development of gene regulation systems (Berens & Hillen, 2004).
+
−
      <br>
+
−
      In our project, tetR was integrated into the design of a modular DNA-stapling system because of its
+
−
      well-characterized behavior, ensuring reliable DNA interactions.
+
 
     </p>
 
     </p>
 
   </section>
 
   </section>
−
   <section id="2.2">
+
   <section id?="2.2">
−
     <h1>2.2mScarlet-I</h1>
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     <h1>2.2 mNeonGreen</h1>
 
     <p>
 
     <p>
−
       mScarlet-I is a rapidly-maturing monomeric red fluorescent protein designed by Bindels <i>et al.</i> in 2016. It
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       mNeonGreen is a bright, monomeric fluorescent protein from <i>Branchiostoma lanceolatum</i> discovered by Shaner
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      is a derivative of the original mScarlet, with a single amino acid substitution (T74I) that enhances its
+
      <i>et al.</i> (2013). It exhibits fast maturation, high photostability, and a high quantum yield. With an
−
       maturation
+
       excitation peak
−
       speed and fluorescence properties (Bindels <i>et al.</i>, 2016). mScarlet-I absorbs at 569 nm and emits at 594 nm.
+
       at 506 nm and an emission maximum at 517 nm, mNeonGreen is ideal for bioimaging applications (Shaner <i>et
−
    </p>
+
        al.</i>,
−
    <p>
+
      2013). Its high quantum yield and stability make it an optimal electron donor for Förster Resonance Energy
−
    <p>
+
      Transfer (FRET). When paired with mScarlet-I, it generates three times the intensity compared to mCherry.
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      mScarlet-I stands out among red fluorescent proteins for its high quantum yield (0.54), long fluorescence lifetime
+
−
      (3.1 ns), and rapid maturation time of approximately 36 minutes. These features ensure strong fluorescence
+
−
      intensity and make it an ideal candidate for Förster Resonance Energy Transfer assays (McCullock <i>et al.</i> 2020). When paired with mNeonGreen, mScarlet-I
+
 
     </p>
 
     </p>
 
   </section>
 
   </section>
 
   <section id="2.3">
 
   <section id="2.3">
−
      <h1>2.3 Förster Resonance Energy Transfer (FRET)</h1>
+
    <h1>2.3 Förster Resonance Energy Transfer (FRET)</h1>
−
      <p>Förster Resonance Energy Transfer (FRET) is a distance-dependent physical process where energy is transferred
+
    <p>Förster Resonance Energy Transfer (FRET) is a distance-dependent physical process where energy is transferred
−
        non-radiatively from an excited donor fluorophore to an acceptor fluorophore via dipole-dipole coupling. The
+
      non-radiatively from an excited donor fluorophore to an acceptor fluorophore via dipole-dipole coupling. The
−
        efficiency of energy transfer is highly sensitive to the distance between the donor and acceptor, typically in
+
      efficiency of energy transfer is highly sensitive to the distance between the donor and acceptor, typically in the
−
        the
+
      range of 1-10 nm, making FRET ideal for studying molecular proximity (Hochreiter <i>et al.</i>, 2019). This
−
        range of 1-10 nm, making FRET ideal for studying molecular proximity (Hochreiter <i>et al.</i>, 2019). This
+
      proximity
−
        proximity
+
      sensitivity is governed by the Förster radius (R₀), which is the distance at which 50% energy transfer occurs.
−
        sensitivity is governed by the Förster radius (R₀), which is the distance at which 50% energy transfer occurs.
+
      Factors affecting FRET efficiency include the overlap of the donor's emission spectrum with the acceptor's
−
        Factors affecting FRET efficiency include the overlap of the donor's emission spectrum with the acceptor's
+
      absorption spectrum, the quantum yield of the donor, and the relative orientation of the fluorophores (Wu & Brand,
−
        absorption spectrum, the quantum yield of the donor, and the relative orientation of the fluorophores (Wu &
+
      1994). These characteristics allow FRET to detect interactions such as protein-DNA binding or DNA proximity in
−
        Brand,
+
      real time.
−
        1994). These characteristics allow FRET to detect interactions such as protein-DNA binding or DNA proximity in
+
      For our assay, we selected mNeonGreen and mScarlet-I as donor and acceptor, respectively, due to their strong
−
        real time.
+
      fluorescence, spectral overlap, and minimal photobleaching, ensuring high FRET efficiency in our system (Bindels
−
        For our assay, we selected mNeonGreen and mScarlet-I as donor and acceptor, respectively, due to their strong
+
      <i>et al.</i>, 2017; Shaner <i>et al.</i>, 2013). FRET's sensitivity to small changes in distance makes it
−
        fluorescence, spectral overlap, and minimal photobleaching, ensuring high FRET efficiency in our system (Bindels
+
      especially powerful
−
        <i>et al.</i>, 2017; Shaner <i>et al.</i>, 2013). FRET's sensitivity to small changes in distance makes it
+
      for analyzing molecular interactions in living cells (Okamoto & Sako, 2017).
−
        especially powerful
+
    </p>
−
        for analyzing molecular interactions in living cells (Okamoto & Sako, 2017).
+
    <div class="thumb">
−
      </p>
+
      <div class="thumbinner" style="width:550px"><img alt=""
−
      <div class="thumb">
+
          src="https://static.igem.wiki/teams/5237/wetlab-results/fpbase-fret-mng-msci.svg" style="width:99%;"
−
        <div class="thumbinner" style="width:550px"><img alt=""
+
          class="thumbimage">
−
            src="https://static.igem.wiki/teams/5237/wetlab-results/fpbase-fret-mng-msci.svg" style="width:99%;"
+
        <div class="thumbcaption">
−
            class="thumbimage">
+
          <i><b>Figure 2: Overview of excitation and emission spectrum of mNeonGreen and m-Scarlet and it's properties
−
          <div class="thumbcaption">
+
              as a FRET pair</b></i>
−
            <i><b>Figure 2: Overview of excitation and emission spectrum of mNeonGreen and m-Scarlet and it's properties
+
−
                as a FRET pair</b></i>
+
−
          </div>
+
 
         </div>
 
         </div>
 +
      </div>
 
       </div>
 
       </div>
 
   </section>
 
   </section>
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   <section id="3">
 
   <section id="3">
 
     <h1>3. Assembly and part evolution</h1>
 
     <h1>3. Assembly and part evolution</h1>
−
     <p></p>
+
     <p>The amino acid sequence of mNeonGreen was taken from the fluorescent protein database (<a
 +
        href="https://www.fpbase.org/">FPbase</a>) and codon optimized for use in <i>E. coli</i>.
 +
      It was fused to thhe N-terminus of Oct1-DBD (<a href="https://parts.igem.org/Part:BBa_K5237004">BBa_K52347004</a>)
 +
      for protein purification of Oct1-DBD and <i>in vivo</i> FRET measurements.
 +
    </p>
 
   </section>
 
   </section>
 
   <section id="4">
 
   <section id="4">
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       </div>
 
       </div>
 
     </div>
 
     </div>
 +
 +
 
   </section>
 
   </section>
 
   <section id="5">
 
   <section id="5">
 
     <h1>5. References</h1>
 
     <h1>5. References</h1>
−
    <p>Wu, W., Zhang, L., Yao, L., Tan, X., Liu, X., & Lu, X. (2015). Genetically assembled fluorescent biosensor
 
−
      for in situ detection of bio-synthesized alkanes. Scientific reports, 5, 10907. <a
 
−
        href="https://doi.org/10.1038/srep10907" target="_blank">https://doi.org/10.1038/srep10907</a></p>
 
−
  </section>
 
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Revision as of 11:45, 29 September 2024

BBa_K5237016

FRET-Donor: mNeonGreen-Oct1

This composite part is a fusion protein of Oct1-DBD and mNeonGreen. It was used as a FRET donor in combination with tetR-mScarlet-I as an acceptor. This part was used to measure the proximity of two DNA strands by FRET fluoresence measurements.

 

The PICasSO Toolbox


Figure 1: Example how the part collection can be used to engineer new staples


The 3D organization of the genome plays a crucial role in regulating gene expression in eukaryotic cells, impacting cellular behavior, evolution, and disease. Beyond the linear DNA sequence, the spatial arrangement of chromatin, influenced by DNA-DNA interactions, shapes pathways of gene regulation. However, the tools to precisely manipulate this genomic architecture remain limited, rendering it challenging to explore the full potential of the 3D genome in synthetic biology. We - iGEM Team Heidelberg 2024 - have developed PICasSO, a powerful molecular toolbox based on various DNA-binding proteins to address this issue.

The PICasSO part collection offers a comprehensive, modular platform for precise manipulation and re-programming of DNA-DNA interactions using protein staples in living cells, enabling researchers to recreate natural 3D genomic interactions, such as enhancer hijacking, or to design entirely new spatial architectures for gene regulation. Beyond its versatility, PICasSO includes robust assay systems to support the engineering, optimization, and testing of new staples, ensuring functionality in vitro and in vivo. We took special care to include parts crucial for testing every step of the cycle (design, build, test, learn) when engineering new parts

At its heart, the PICasSO part collection consists of three categories. (i) Our DNA-binding proteins include our finalized enhancer hijacking Cas staple as well as half staples that can be used by scientists to compose entirely new Cas staples in the future. We also include our simple staples that serve as controls for successful stapling and can be further engineered to create alternative, simpler and more compact staples. (ii) As functional elements, we list additional parts that enhance the functionality of our Cas and Basic staples. These consist of protease-cleavable peptide linkers and inteins that allow condition-specific, dynamic stapling in vivo. Besides staple functionality, we also include the parts to enable the efficient delivery of PICasSO's with our interkingdom conjugation system.

(iii) As the final component of our collection, we provide parts that support the use of our custom readout systems. These include components of our established FRET-based proximity assay system, enabling users to confirm accurate stapling. Additionally, we offer a complementary, application-oriented testing system for functional readout via a luciferase reporter, which allows for straightforward experimental simulation of enhancer hijacking.

The following table gives a complete overview of all parts in our PICasSO toolbox. The highlighted parts showed exceptional performance as described on our iGEM wiki and can serve as a reference. The other parts in the collection are versatile building blocks designed to provide future iGEMers with the flexibility to engineer their own custom Cas staples, enabling further optimization and innovation

Our part collection includes:

DNA-binding proteins: The building blocks for engineering of custom staples for DNA-DNA interactions with a modular system ensuring easy assembly.
BBa_K5237000 fgRNA Entryvector MbCas12a-SpCas9 Entryvector for simple fgRNA cloning via SapI
BBa_K5237001 Staple subunit: dMbCas12a-Nucleoplasmin NLS Staple subunit that can be combined to form a functional staple, for example with fgRNA and dCas9
BBa_K5237002 Staple subunit: SV40 NLS-dSpCas9-SV40 NLS Staple subunit that can be combined to form a functional staple, for example with our fgRNA or dCas12a
BBa_K5237003 Cas-Staple: SV40 NLS-dMbCas12a-dSpCas9-Nucleoplasmin NLS Functional Cas staple that can be combined with sgRNA or fgRNA to bring two DNA strands in close proximity
BBa_K5237004 Staple subunit: Oct1-DBD Staple subunit that can be combined to form a functional staple, for example with TetR.
Can also be combined with a fluorescent protein as part of the FRET proximity assay
BBa_K5237005 Staple subunit: TetR Staple subunit that can be combined to form a functional staple, for example with Oct1.
Can also be combined with a fluorescent protein as part of the FRET proximity assay
BBa_K5237006 Simple taple: TetR-Oct1 Functional staple that can be used to bring two DNA strands in close proximity
BBa_K5237007 Staple subunit: GCN4 Staple subunit that can be combined to form a functional staple, for example with rGCN4
BBa_K5237008 Staple subunit: rGCN4 Staple subunit that can be combined to form a functional staple, for example with rGCN4
BBa_K5237009 Mini staple: bGCN4 Assembled staple with minimal size that can be further engineered
Functional elements: Protease cleavable peptide linkers and inteins are used to control and modify staples for further optimization for custom applications.
BBa_K5237010 Cathepsin B-Cleavable Linker (GFLG) Cathepsin B cleavable peptide linker, that can be used to combine two staple subunits ,to make responsive staples
BBa_K5237011 Cathepsin B Expression Cassette Cathepsin B which can be selectively express to cut the cleavable linker
BBa_K5237012 Caged NpuN Intein Undergoes protein transsplicing after protease activation, can be used to create functionalized staple units
BBa_K5237013 Caged NpuC Intein Undergoes protein transsplicing after protease activation, can be used to create functionalized staple units
BBa_K5237014 fgRNA processing casette Processing casette to produce multiple fgRNAs from one transcript, can be used for multiplexing
BBa_K5237015 Intimin anti-EGFR Nanobody Interkindom conjugation between bacteria and mammalian cells, as alternative delivery tool for large constructs
Readout Systems: FRET and enhancer recruitment to measure proximity of stapled DNA in bacterial and mammalian living cells enabling swift testing and easy development for new systems.
BBa_K5237016 FRET-Donor: mNeonGreen-Oct1 Donor part for the FRET assay binding the Oct1 binding cassette. Can be used to visualize DNA-DNA proximity
BBa_K5237017 FRET-Acceptor: TetR-mScarlet-I Acceptor part for the FRET assay binding the TetR binding cassette. Can be used to visualize DNA-DNA proximity
BBa_K5237018 Oct1 Binding Casette DNA sequence containing 12 Oct1 binding motifs, can be used for different assays such as the FRET proximity assay
BBa_K5237019 TetR Binding Cassette DNA sequence containing 12 Oct1 binding motifs, can be used for different assays such as the FRET proximity assay
BBa_K5237020 Cathepsin B-Cleavable Trans-Activator: NLS-Gal4-GFLG-VP64 Readout system that responds to protease activity. It was used to test Cathepsin-B cleavable linker.
BBa_K5237021 NLS-Gal4-VP64 Trans-activating enhancer, that can be used to simulate enhancer hijacking.
BBa_K5237022 mCherry Expression Cassette: UAS, minimal Promotor, mCherry Readout system for enhancer binding. It was used to test Cathepsin-B cleavable linker.
BBa_K5237023 Oct1 - 5x UAS binding casette Oct1 and UAS binding cassette, that was used for the simulated enhancer hijacking assay.
BBa_K5237024 TRE-minimal promoter- firefly luciferase Contains Firefly luciferase controlled by a minimal promoter. It was used as a luminescence readout for simulated enhancer hijacking.

1. Sequence overview

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 710
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

2. Usage and Biology

2.1 Oct1-DBD

Oct1-DBD is the DNA-binding domain of the human transcription factor Oct1 (POU2F1), which plays a key role in gene regulation, immune response, and stress adaptation in eukaryotic cells. This domain specifically binds to the octamer motif (5'-ATGCAAAT-3') within promoter and enhancer regions, influencing transcriptional activity (Lundbäck et al., 2000). The Oct1-DBD consists of both a POU-specific domain and a POU homeodomain, which work together to form a stable complex with DNA (Park et al., 2013, Stepchenko et al. 2021).

In synthetic biology, Oct1-DBD was previously used for plasmid display technology due to its strong binding affinity (KD = 9 × 10-11 M). Proteins fused with Oct1-DBD showed increased expression and protein solubility (Parker et al. 2020).

2.2 mNeonGreen

mNeonGreen is a bright, monomeric fluorescent protein from Branchiostoma lanceolatum discovered by Shaner et al. (2013). It exhibits fast maturation, high photostability, and a high quantum yield. With an excitation peak at 506 nm and an emission maximum at 517 nm, mNeonGreen is ideal for bioimaging applications (Shaner et al., 2013). Its high quantum yield and stability make it an optimal electron donor for Förster Resonance Energy Transfer (FRET). When paired with mScarlet-I, it generates three times the intensity compared to mCherry.

2.3 Förster Resonance Energy Transfer (FRET)

Förster Resonance Energy Transfer (FRET) is a distance-dependent physical process where energy is transferred non-radiatively from an excited donor fluorophore to an acceptor fluorophore via dipole-dipole coupling. The efficiency of energy transfer is highly sensitive to the distance between the donor and acceptor, typically in the range of 1-10 nm, making FRET ideal for studying molecular proximity (Hochreiter et al., 2019). This proximity sensitivity is governed by the Förster radius (R₀), which is the distance at which 50% energy transfer occurs. Factors affecting FRET efficiency include the overlap of the donor's emission spectrum with the acceptor's absorption spectrum, the quantum yield of the donor, and the relative orientation of the fluorophores (Wu & Brand, 1994). These characteristics allow FRET to detect interactions such as protein-DNA binding or DNA proximity in real time. For our assay, we selected mNeonGreen and mScarlet-I as donor and acceptor, respectively, due to their strong fluorescence, spectral overlap, and minimal photobleaching, ensuring high FRET efficiency in our system (Bindels et al., 2017; Shaner et al., 2013). FRET's sensitivity to small changes in distance makes it especially powerful for analyzing molecular interactions in living cells (Okamoto & Sako, 2017).

Figure 2: Overview of excitation and emission spectrum of mNeonGreen and m-Scarlet and it's properties as a FRET pair

3. Assembly and part evolution

The amino acid sequence of mNeonGreen was taken from the fluorescent protein database (FPbase) and codon optimized for use in E. coli. It was fused to thhe N-terminus of Oct1-DBD (BBa_K52347004) for protein purification of Oct1-DBD and in vivo FRET measurements.

4. Results

The FRET assay was developed using a two-plasmid system in bacterial cells. The expression plasmid contains a tetR binding site (BBa_K5237019) and expresses three key proteins under the control of a single T7 promoter in a polycistronic operon: (1) tetR-Oct1, our simple staple fusion protein that acts as a bivalent DNA-binding protein, tethering two plasmids via tetR and Oct1 binding sites; (2) Oct1-mNeonGreen, serving as the FRET donor; and (3) tetR-mScarlet-I, the FRET acceptor. This ensures all three proteins are co-expressed in similar stoichiometry. The folding plasmid contains an Oct1 binding site (BBa_K5237018) for the staple and FRET donor binding.

When tetR-Oct1 binds its respective sites on both plasmids, it brings mNeonGreen and mScarlet-I into close proximity, facilitating FRET between the two fluorophores. Successful stapling of the plasmids results in increased energy transfer from mNeonGreen to mScarlet-I, which can be detected by exciting mNeonGreen and measuring enhanced emission from mScarlet-I. A positive control, consisting of a direct fusion of mNeonGreen and mScarlet-I, ensures maximal FRET efficiency and serves as a benchmark for the assay.

Fluorescence intensity, normalized to cell count, of mNeonGreen and mScarlet-I was measured 18 h after induction with varying IPTG concentration (Figure 3). An increasing expression strength is visible for decreasing IPTG concentrations. Fluorescence intensity of the positive control was significantly stronger compared to the negative control and staple. The negative control and staple, which both have the same expression plasmid construct, had similar fluorescence intensity for mNeonGreen and mScarlet-I down to approximately 0.05 mM. Lower concentrations resulted in strong discrepancies. To ensure comparability between the negative control and staple, further fluorescence intensity measurements were conducted after induction with 0.05 mM IPTG. Fluorescence measurement of the donor and acceptor showed similar intensities, with only a small significant difference for mNeonGreen. A large difference could be measured between the staple and negative control, indicating proximity induced FRET. (Figure 3)

pic
Figure 3: Fluorescence of mNeonGreen, mScarlet-I and FRET. Fluorescence intensity of mNeonGreen (ex. 490 nm, em. 530 nm), mScarlet-I (ex. 560 nm, em. 600 nm), and FRET (ex. 490 nm, em. 600 nm) was measured 18 h after induction with IPTG and normalized to cell count (OD600). A), B) Fluorescence intensity of mNeonGreen and mScarlet-I after induction with different IPTG concentrations. C) Fluorescence intensity of FRET pair induced with 0.025 mM IPTG. Statistical significance was tested for with Ordinary two-way ANOVA with Šidák's multiple comparison test, with a single pooled variance. *p < 0.05, ****p < 0.001. Only significant results, within groups are shown. Data is depcited as mean ± SD.

5. References