Difference between revisions of "Part:BBa K5237007"
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       <h2>4.1 Protein expression and purification</h2>
 
       <h2>4.1 Protein expression and purification</h2>
 
       <p>The FLAG-GCN4 protein could be readily expressed in <i>E. coli</i> BL21 (DE3). The protein was purified using an anti-FLAG resin.
 
       <p>The FLAG-GCN4 protein could be readily expressed in <i>E. coli</i> BL21 (DE3). The protein was purified using an anti-FLAG resin.
−
         Fractions taken during purification were analyzed by SDS-PAGE.
+
         Fractions taken during purification were analyzed by SDS-PAGE. Purified protein was quantified using a Lowry assay, 1.18 mg/mL were obtained, resulting in 153 µM of monomeric FLAG-GCN4.
 
       </p>
 
       </p>
 
       <div class="thumb">
 
       <div class="thumb">
Line 348: Line 348:
 
       </div>
 
       </div>
 
     </section>
 
     </section>
 +
    <section id="4.2">
 +
      <h2>4.2 Electrophoretic Mobility shift assay</h2>
 +
      <p>
 +
        GCN4 was tested for its DNA-binding capabilities using an electrophoretic mobility shift assay (EMSA). The protein was incubated with a DNA probe containing the GCN4 binding site. The formation of a protein-DNA complex was analyzed by native PAGE.
 +
        To further analyze DNA binding, quantitative shift assays were performed for GCN4 and rGCN4 (<a href="https://parts.igem.org/Part:BBa:K5237008">BBa_K5237008</a>).
 +
        0.5 µM DNA were incubated with varying concentrations of protein until equilibration. After
 +
        electrophoresis, bands were stained with SYBR-Safe and quantified based on pixel intensity. The
 +
        obtained values were fitted to equation 1, describing formation of a 2:1 protein-DNA complex:
 +
        <br><br>
 +
        Θ<sub>app</sub> = Θ<sub>min</sub> + (Θ<sub>max</sub> - Θ<sub>min</sub>) &#215;
 +
        (K<sub>a</sub><sup>2</sup> [L]<sub>tot</sub><sup>2</sup>) / (1 + K<sub>a</sub><sup>2</sup>
 +
        [L]<sub>tot</sub><sup>2</sup>)
 +
        <span style="float: right;">Equation 1</span>
 +
        <br><br>
 +
        Here [L]<sub>tot</sub> describes the total protein monomer concentration, K<sub>a</sub>
 +
        corresponds
 +
        to the apparent monomeric equilibration constant. The Θ<sub>min/max</sub> values are the
 +
        experimentally
 +
        determined site saturation values (For this experiment 0 and 1 were chosen for min and max
 +
        respectively). GCN4 binds to its optimal DNA binding motif with an apparent dissociation
 +
        constant K<sub>k</sub> of (0.2930.033)&#215;10<sup>-6</sup> M, which is almost identical to the
 +
        rGCN4 binding
 +
        affinity to INVii a <sub>d</sub> of (0.2980.030)&#215;10<sup>-6</sup> M.
 +
      </p>
 +
      <div class="thumb">
 +
        <div class="thumbinner" style="width:500px">
 +
          <img alt="" src="https://static.igem.wiki/teams/5237/wetlab-results/mist-leucine-zipper-kd-plot.svg" style="width:99%;" class="thumbimage">
 +
          <div class="thumbcaption">
 +
            <i><b>Figure 3: Quantitative EMSA</b>Quantitative assessment of binding affinity for GCN4 and rGCN4. Proteins of
 +
              different concentrations were incubated with 0.5 µM DNA until equilibrium and fraction bound analyzed, after gel electrophoresis, by dividing pixel intensity of
 +
              bound fraction with pixel intensity of bound and unbound fraction. At least three separate measurements were conducted
 +
              for each data point. Values are presented as mean +/- SD.</i>
 +
          </div>
 +
      </div>
 +
      <p>
 +
        The apparent binding kinetics calculated for GCN4 ((0.2930.033) &#215; 10<sup>-6</sup> M) and rGCN4
 +
        ((0.2980.030) &#215; 10<sup>-6</sup> M) are
 +
        approximately a factor 10 higher then those described in literature ((96) &#215; 10<sup>-8</sup> M for
 +
        GCN4 and (2.90.8) &#215; 10<sup>-8</sup> M for rGCN4) (Hollenbeck <i class="italic">et al.</i>, 2001). The
 +
        differences could be due to the lower sensitivity of SYBR-Safe
 +
        staining compared to radio-labeled oligos.
 +
        <br><br>
 +
        The FLAG-tag fusion to the N-terminus of proteins could potentially decrease binding affinity, likely due
 +
        to steric hindrance affecting the interaction with DNA. Interestingly, the differences in binding affinity
 +
        between GCN4 and rGCN4 appear negligible. Since GCN4 binds to DNA via its N-terminus and rGCN4 binds
 +
        C-terminally, the FLAG-tag likely does not directly influence DNA binding. However, it may influence the
 +
        dimerization of the proteins, which is necessary for DNA binding. To further investigate this, the
 +
        FLAG-tag can be cleaved using an enterokinase and potential changes in binding affinity analyzed
 +
      </p>
 
   </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
+
     <p>
−
      for in situ detection of bio-synthesized alkanes. Scientific reports, 5, 10907. <a
+
      Fried, M. G. (1989). Measurement of protein-DNA interaction parameters by electrophoresis mobility shift assay.
−
        href="https://doi.org/10.1038/srep10907" target="_blank">https://doi.org/10.1038/srep10907</a></p>
+
      <i>Electrophoresis, 10</i>(5–6), 366–376.
 +
      <a href="https://doi.org/10.1002/elps.1150100515" target="_blank">https://doi.org/10.1002/elps.1150100515</a>
 +
    </p>
 +
    <p>
 +
      Hellman, L. M., & Fried, M. G. (2007). Electrophoretic mobility shift assay (EMSA) for detecting protein-nucleic acid interactions.
 +
      <i>Nature Protocols, 2</i>(8), 1849–1861.
 +
      <a href="https://doi.org/10.1038/nprot.2007.249" target="_blank">https://doi.org/10.1038/nprot.2007.249</a>
 +
    </p>
 +
    <p>
 +
      Hollenbeck, J. J., & Oakley, M. G. (2000). GCN4 binds with high affinity to DNA sequences containing a single consensus half-site.
 +
      <i>Biochemistry, 39</i>(21), 6380–6389.
 +
      <a href="https://doi.org/10.1021/bi992705n" target="_blank">https://doi.org/10.1021/bi992705n</a>
 +
    </p>
 +
    <p>
 +
      Hollenbeck, J. J., Gurnon, D. G., Fazio, G. C., Carlson, J. J., & Oakley, M. G. (2001). A GCN4 variant with a C-terminal basic region binds to DNA with wild-type affinity.
 +
      <i>Biochemistry, 40</i>(46), 13833–13839.
 +
      <a href="https://doi.org/10.1021/bi0106916" target="_blank">https://doi.org/10.1021/bi0106916</a>
 +
    </p>
 +
    <p>
 +
      McKnight, S. L., & Tjian, R. (1988). Analysis of transcriptional regulatory proteins of the human genome.
 +
      <i>Science, 241</i>(4870), 1306–1313.
 +
      <a href="https://doi.org/10.1126/science.2847199" target="_blank">https://doi.org/10.1126/science.2847199</a>
 +
    </p>
 +
    <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.  
 +
      <i>Scientific Reports, 5</i>, 10907.  
 +
      <a href="https://doi.org/10.1038/srep10907" target="_blank">https://doi.org/10.1038/srep10907</a>
 +
    </p>
 
   </section>
 
   </section>
 +
 
 
</body>
 
</body>
  
 
</html>
 
</html>

Revision as of 20:48, 28 September 2024


BBa_K5237007

Staple subunit: GCN4

GCN4 is a yeast transcription factor belonging to the bZip family of DNA-binding proteins. It consists of a basic region and a leucine zipper dimerization domain, binding DNA as a homodimer via its N-terminal region

 

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 parts 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
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

2. Usage and Biology

GCN4 is a yeast transcription factor from the bZip family of DNA-binding proteins, first discovered by McKnight and co-workers in 1988. The bZip motif features a coiled-coil leucine zipper dimerization domain paired with a highly charged basic region, which directly interacts with DNA. GCN4 binds specifically to the cyclic AMP response element (CRE) in the promoter regions of target genes, primarily through its basic residues at the N-terminus.

In our project, GCN4 was employed to study DNA-binding kinetics and develop a minimal "Mini staple" that brings two DNA target sites into proximity by binding them simultaneously. This "Mini staple" was designed as a versatile tool for precise DNA manipulation in synthetic biology applications.

The DNA-binding properties of GCN4 were tested using an electrophoretic mobility shift assay (EMSA) to quantify binding affinity and kinetics. EMSA is a widely adopted method to study DNA-protein interactions. It works on the principle that nucleic acids bound to proteins exhibit reduced electrophoretic mobility compared to unbound nucleic acids (Hellman & Fried, 2007). EMSA can be employed both qualitatively, to assess DNA-binding capabilities, and quantitatively, to determine critical parameters such as binding stoichiometry and the apparent dissociation constant (Kd) (Fried, 1989).

3. Assembly and part evolution

The GCN4 amino acid sequence was taken from literature (Hollenbeck & Oakley 1999) and codon optimized for E. coli. A FLAG-tag (DYKDDDDK) was added to the N-terminus for protein purification. The FLAG-tag can be cleaved off using an Enterokinase, if necessary. The FLAG-GCN4 sequence was cloned into a T7 expression vector and expressed using E. coli BL21 (DE3) cells.

4. Results

4.1 Protein expression and purification

The FLAG-GCN4 protein could be readily expressed in E. coli BL21 (DE3). The protein was purified using an anti-FLAG resin. Fractions taken during purification were analyzed by SDS-PAGE. Purified protein was quantified using a Lowry assay, 1.18 mg/mL were obtained, resulting in 153 µM of monomeric FLAG-GCN4.

Figure 2: SDS-PAGE analysis of FLAG-GCN4 purification Fractions analysed are the raw lysate, flow through and eluate. Depicted is GCN4 (this part), rGCN4 (BBa_K5237008), and bGCN4 (BBa_K5237009). Protein size is indicated next to construct name and purified band with protein of interest highlighted by a red box.

4.2 Electrophoretic Mobility shift assay

GCN4 was tested for its DNA-binding capabilities using an electrophoretic mobility shift assay (EMSA). The protein was incubated with a DNA probe containing the GCN4 binding site. The formation of a protein-DNA complex was analyzed by native PAGE. To further analyze DNA binding, quantitative shift assays were performed for GCN4 and rGCN4 (BBa_K5237008). 0.5 µM DNA were incubated with varying concentrations of protein until equilibration. After electrophoresis, bands were stained with SYBR-Safe and quantified based on pixel intensity. The obtained values were fitted to equation 1, describing formation of a 2:1 protein-DNA complex:

Θapp = Θmin + (Θmax - Θmin) × (Ka2 [L]tot2) / (1 + Ka2 [L]tot2) Equation 1

Here [L]tot describes the total protein monomer concentration, Ka corresponds to the apparent monomeric equilibration constant. The Θmin/max values are the experimentally determined site saturation values (For this experiment 0 and 1 were chosen for min and max respectively). GCN4 binds to its optimal DNA binding motif with an apparent dissociation constant Kk of (0.2930.033)×10-6 M, which is almost identical to the rGCN4 binding affinity to INVii a d of (0.2980.030)×10-6 M.

Figure 3: Quantitative EMSAQuantitative assessment of binding affinity for GCN4 and rGCN4. Proteins of different concentrations were incubated with 0.5 µM DNA until equilibrium and fraction bound analyzed, after gel electrophoresis, by dividing pixel intensity of bound fraction with pixel intensity of bound and unbound fraction. At least three separate measurements were conducted for each data point. Values are presented as mean +/- SD.

The apparent binding kinetics calculated for GCN4 ((0.2930.033) × 10-6 M) and rGCN4 ((0.2980.030) × 10-6 M) are approximately a factor 10 higher then those described in literature ((96) × 10-8 M for GCN4 and (2.90.8) × 10-8 M for rGCN4) (Hollenbeck et al., 2001). The differences could be due to the lower sensitivity of SYBR-Safe staining compared to radio-labeled oligos.

The FLAG-tag fusion to the N-terminus of proteins could potentially decrease binding affinity, likely due to steric hindrance affecting the interaction with DNA. Interestingly, the differences in binding affinity between GCN4 and rGCN4 appear negligible. Since GCN4 binds to DNA via its N-terminus and rGCN4 binds C-terminally, the FLAG-tag likely does not directly influence DNA binding. However, it may influence the dimerization of the proteins, which is necessary for DNA binding. To further investigate this, the FLAG-tag can be cleaved using an enterokinase and potential changes in binding affinity analyzed

5. References

Fried, M. G. (1989). Measurement of protein-DNA interaction parameters by electrophoresis mobility shift assay. Electrophoresis, 10(5–6), 366–376. https://doi.org/10.1002/elps.1150100515

Hellman, L. M., & Fried, M. G. (2007). Electrophoretic mobility shift assay (EMSA) for detecting protein-nucleic acid interactions. Nature Protocols, 2(8), 1849–1861. https://doi.org/10.1038/nprot.2007.249

Hollenbeck, J. J., & Oakley, M. G. (2000). GCN4 binds with high affinity to DNA sequences containing a single consensus half-site. Biochemistry, 39(21), 6380–6389. https://doi.org/10.1021/bi992705n

Hollenbeck, J. J., Gurnon, D. G., Fazio, G. C., Carlson, J. J., & Oakley, M. G. (2001). A GCN4 variant with a C-terminal basic region binds to DNA with wild-type affinity. Biochemistry, 40(46), 13833–13839. https://doi.org/10.1021/bi0106916

McKnight, S. L., & Tjian, R. (1988). Analysis of transcriptional regulatory proteins of the human genome. Science, 241(4870), 1306–1313. https://doi.org/10.1126/science.2847199

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. https://doi.org/10.1038/srep10907