Difference between revisions of "Part:BBa K5107008"

 
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<partinfo>BBa_K5107003 short</partinfo>
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<partinfo>BBa_K5107008 short</partinfo>
  
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ERE 5 times
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T7-EREmin-sB-T is a construct used by the cell free biosensor.The EREmin is recognised by the steroid estrogen hormone receptor. Then in vitro transcription of the momomeric broccoli is taken place if the hormone is present or not in the cell free solution.
  
−
<!-- Add more about the biology of this part here
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==Usage and Biology==
−
===Usage and Biology===
+
For the structure of the biosensor, we took inspiration from the ROSALIND cell-free biosensor<html><a href="#ref1">[1]</a></html>, modifying their design to match our goals. We kept the general idea of having a Transcription Factor (TF) altering the activity of a RNA polymerase, and the output signal as a consequence. We tailored the ROSALIND concept by selecting specific custom transcription factors (TFs) as receptors and designing unique operator sequences to serve as responsive elements. The exact parts are described here:
 +
 
 +
<center><html><img src="https://static.igem.wiki/teams/5107/eng-parts-re.webp"width="600" height="200">
 +
<figcaption><b>Figure 1:</b> Overview of the T7-HRE-sB-T and T7-ERE-sB-T DNA fragments used for the cell-free system. T7 promoter, response elements, aptamer parts and terminators are shown. Not to scale.</figcaption></html></center>.
 +
 
 +
==Cell free biosensor==
 +
 
 +
<html>
 +
<center>
 +
    <div style="display: flex; justify-content: center;">
 +
        <figure>
 +
            <img src="https://static.igem.wiki/teams/5107/eng-cellfreesystem-p1.webp" height="300" alt="Cell-Free System Part 1">
 +
            <figcaption><b>Figure 2:</b> Cell-Free System - No Hormone/EDC in the environment.</figcaption>
 +
        </figure>
 +
        <figure style="margin-left: 20px;">
 +
            <img src="https://static.igem.wiki/teams/5107/eng-cellfreesystem-p2.webp" height="300" alt="Cell-Free System Part 2">
 +
            <figcaption><b>Figure 3:</b> Cell-Free System - Hormone/EDC in the environment.</figcaption>
 +
        </figure>
 +
    </div>
 +
</center>
 +
</html>
 +
 
 +
==Assemply==
 +
 
 +
{| class="wikitable"
 +
|+ Human Receptor Information
 +
|-
 +
! Human Receptor !! Response Element (Operator Site) !! Natural Hormone !! Plasmid Name (In-Cell System)
 +
|-
 +
| Estrogen Receptor α (ERα) || ERE || 17β-Estradiol || pRR-ERalpha-5Z
 +
|-
 +
| Estrogen Receptor β (ERβ) || ERE || 17β-Estradiol || pRR-ERbeta-5Z
 +
|-
 +
| Glucocorticoid Receptor (GR) || HRE || Dexamethasone || pRR-GR-5Z
 +
|-
 +
| Androgen Receptor (AR) || HRE || Testosterone || pRR-AR-5Z
 +
|-
 +
| Mineralocorticoid Receptor (MR) || HRE || Aldosterone || pRR-MR-5Z
 +
|-
 +
| Progesterone Receptor (PR) || HRE || Progesterone || pRR-PR-5Z
 +
|}
 +
''Table 1: Human Receptor Information''
 +
 
 +
 
 +
 
 +
We designed DNA templates using two response elements: ERE and HRE, designed to interact with the above-listed receptors (see Table 1 for details). We designed versions with only a single response element and only a single repeat. For its minimalistic approach, we denoted these parts <strong>HREminimal</strong> and <strong> HREminimal</strong>. The sequence for these parts can be seen below:
 +
 
 +
{| class="wikitable"
 +
|+ Minimal Response Elements
 +
|-
 +
! Element !! Sequence
 +
|-
 +
| EREminimal || '''CCAGGTCAGAGTGACCTG'''
 +
|-
 +
| HREminimal || '''AGAACAGAGTGTTCT'''
 +
|}
 +
''Table 2: Minimal Response Elements''
 +
 
 +
 
 +
The devices incorporating these parts were named <strong>T7-HREmin-sB-T</strong> and <strong>T7-EREmin-sB-T</strong> for the HREminimal and EREminimal respectively.
 +
 
 +
*Design - Generating a measurable readout
 +
 
 +
As for the output, we decided to go for a less tedious reporter than the one previously used. For our cell-free system, the simplest and fastest way to have an output is to use an aptamer, and we opted for the fluorescent broccoli aptamer, which, when bound to the fluorophore DFHBI-1T, will produce green fluorescence upon excitation.
 +
We design the single broccoli aptamer (abbreviated “<strong>sB</strong>”) surrounded by two tRNA scaffolds to increase its stability (inspired from [[BBa_K3380101]]) in combination with the HREminimal and EREminimal responsive elements, in the parts T7-HREmin-sB-T and T7-EREmin-sB-T.
 +
The reason for this choice is that we discovered a synthesis limitation during a preliminary check on our sponsor IDT's website.
 +
 
 +
The ready-to-be-synthesized DNA templates named <strong>T7-HREminimal-sB-T</strong> and <strong>T7-EREminimal-sB-T</strong>, carrying only one single binding site and the single Broccoli aptamer flanked by two scaffold, are shown in the image 1.
 +
 
 +
 
 +
*Validation
 +
 
 +
The final ROSALIND templates (T7-HRE-sB-T and T7_ERE-sB-T) were obtained by PCR of the target sequences containing only the DNA parts necessary for Cell-Free Transcription System.Here it is shown only the gel electrophoresis of the T7-HRE-sB-T.
 +
<html>
 +
<center>
 +
    <figure>
 +
        <img src="https://static.igem.wiki/teams/5107/parts/pcr-hremin.webp" height="300" alt="">
 +
        <figcaption><b>Figure 2:</b>PCR validation of the cell free biosensor template(T7-HRE-sB-T)..</figcaption>
 +
    </figure>
 +
</center>
 +
</html>
 +
 
 +
 
 +
==Test and Optimization==
 +
 
 +
To test the created parts, we performed two iterations. Firstly, we tested and optimized the fluorescence output without the presence of any receptor or ligand (Test & Learn I), to ensure that the design at its basic level works properly.
 +
Secondly, we proceeded by testing the biosensor on its whole with the receptor and the ligands (Test & Learn II).
 +
 
 +
1. '''Wavelength and Plate reader setting'''
 +
*Rationale: As the signal for the first experiments was erratic and sometimes incoherent, we tried to improve the reading settings.
 +
 
 +
*Result:Higher fluorescence output was yielded by: 
 +
Using the wavelength couple 488/530 nm.
 +
Reading from the top (instead from the bottom)
 +
 
 +
<html>
 +
<center>
 +
    <figure>
 +
        <img src="https://static.igem.wiki/teams/5107/parts/wavelength-comparison-dark.webp" height="300" alt="">
 +
        <figcaption><b>Figure 5:</b> Wavelength optimization.Fluorescein sodium salt was used as reference.</figcaption>
 +
    </figure>
 +
</center>
 +
</html>
 +
2. '''Buffer test'''
 +
*Rationale: Optimize the reaction to increase the signal
 +
 
 +
Initially, for the first transcription test, we used a custom In Vitro Transcription (IVT) buffer recommended from the ROSALIND protocol (where we took the inspiration for the cell-free system). However, we didn’t get any fluorescence emission by using that custom buffer.
 +
 
 +
*Result: The commercial In Vitro Transcription (IVT) buffer was better than the custom made.
 +
When using the commercial buffer, we could see a much higher output signal. The custom buffer clearly is not ideal for the cell-free transcription, while the commercial buffer seems to work much better. A possible explanation for this, is the ionic concentration, which is much higher in the custom buffer (especially NaCl). Either the indicated concentrations were wrong (we in fact acknowledge a mistake in the ROSALIND protocol, as the indicated concentration of the NaCl ion was too high) or we made a mistake in the process of preparing it.
 +
 
 +
3. '''DNA concentration'''
 +
*Rationale: Increase the sensitivity of the biosensor, enlarge the limits of detection, and reduce the cost of testing.
 +
Reducing the amount of DNA enhances both sensitivity and the limit of detection, which are critical factors for our stakeholders. This reduction will ultimately lower the detection threshold, allowing even trace amounts of EDCs in the tested water to produce measurable inhibition. Additionally, it enables us to minimize the use of the receptor, the most expensive component of the biosensor system.
 +
*Results: A concentration of 10 nM of DNA was sufficient to yield a substantial signal and the best one to perform the next experiments, also according to the Modeling analysis.
 +
 
 +
<html>
 +
<center>
 +
    <figure>
 +
        <img src="https://static.igem.wiki/teams/5107/parts/dna-comparison-narrow-dark.webp"  height="300"alt="">
 +
        <figcaption><b>Figure 6:</b> Test for different DNA concentrations in order to have a readable and measurable signal. Measurements were taken after 1 hour for 20 minutes. Error bars represent standard deviation of triplicates.</figcaption>
 +
    </figure>
 +
</center>
 +
</html>
 +
 
 +
 
 +
===Sequence and Features===
 +
 
 +
<partinfo>BBa_K5107008 SequenceandFeatures</partinfo>
  
−
<!-- -->
 
−
<span class='h3bb'>Sequence and Features</span>
 
−
<partinfo>BBa_K5107003 SequenceAndFeatures</partinfo>
 
  
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===
−
<partinfo>BBa_K5107003 parameters</partinfo>
+
<partinfo>BBa_K5107008 parameters</partinfo>
 
<!-- -->
 
<!-- -->
 +
 +
===References===
 +
<html>
 +
<ol>
 +
    <li id="ref1">
 +
        Chen, R., Cheng, H., Jin, P., Song, L., Yue, T., Hull, M., & Mansell, T. J. (2020). 
 +
        <i>Nature Biotechnology</i>, <i>38</i>(10), 1107–1112. https://doi.org/10.1038/s41587-020-0571-7
 +
    </li>
 +
</ol>
 +
</html>

Revision as of 20:40, 29 September 2024


T7-EREmin-sB-T

T7-EREmin-sB-T is a construct used by the cell free biosensor.The EREmin is recognised by the steroid estrogen hormone receptor. Then in vitro transcription of the momomeric broccoli is taken place if the hormone is present or not in the cell free solution.

Usage and Biology

For the structure of the biosensor, we took inspiration from the ROSALIND cell-free biosensor[1], modifying their design to match our goals. We kept the general idea of having a Transcription Factor (TF) altering the activity of a RNA polymerase, and the output signal as a consequence. We tailored the ROSALIND concept by selecting specific custom transcription factors (TFs) as receptors and designing unique operator sequences to serve as responsive elements. The exact parts are described here:

Figure 1: Overview of the T7-HRE-sB-T and T7-ERE-sB-T DNA fragments used for the cell-free system. T7 promoter, response elements, aptamer parts and terminators are shown. Not to scale.
.

Cell free biosensor

Cell-Free System Part 1
Figure 2: Cell-Free System - No Hormone/EDC in the environment.
Cell-Free System Part 2
Figure 3: Cell-Free System - Hormone/EDC in the environment.

Assemply

Human Receptor Information
Human Receptor Response Element (Operator Site) Natural Hormone Plasmid Name (In-Cell System)
Estrogen Receptor α (ERα) ERE 17β-Estradiol pRR-ERalpha-5Z
Estrogen Receptor β (ERβ) ERE 17β-Estradiol pRR-ERbeta-5Z
Glucocorticoid Receptor (GR) HRE Dexamethasone pRR-GR-5Z
Androgen Receptor (AR) HRE Testosterone pRR-AR-5Z
Mineralocorticoid Receptor (MR) HRE Aldosterone pRR-MR-5Z
Progesterone Receptor (PR) HRE Progesterone pRR-PR-5Z

Table 1: Human Receptor Information


We designed DNA templates using two response elements: ERE and HRE, designed to interact with the above-listed receptors (see Table 1 for details). We designed versions with only a single response element and only a single repeat. For its minimalistic approach, we denoted these parts HREminimal and HREminimal. The sequence for these parts can be seen below:

Minimal Response Elements
Element Sequence
EREminimal CCAGGTCAGAGTGACCTG
HREminimal AGAACAGAGTGTTCT

Table 2: Minimal Response Elements


The devices incorporating these parts were named T7-HREmin-sB-T and T7-EREmin-sB-T for the HREminimal and EREminimal respectively.

  • Design - Generating a measurable readout

As for the output, we decided to go for a less tedious reporter than the one previously used. For our cell-free system, the simplest and fastest way to have an output is to use an aptamer, and we opted for the fluorescent broccoli aptamer, which, when bound to the fluorophore DFHBI-1T, will produce green fluorescence upon excitation. We design the single broccoli aptamer (abbreviated “sB”) surrounded by two tRNA scaffolds to increase its stability (inspired from BBa_K3380101) in combination with the HREminimal and EREminimal responsive elements, in the parts T7-HREmin-sB-T and T7-EREmin-sB-T. The reason for this choice is that we discovered a synthesis limitation during a preliminary check on our sponsor IDT's website.

The ready-to-be-synthesized DNA templates named T7-HREminimal-sB-T and T7-EREminimal-sB-T, carrying only one single binding site and the single Broccoli aptamer flanked by two scaffold, are shown in the image 1.


  • Validation

The final ROSALIND templates (T7-HRE-sB-T and T7_ERE-sB-T) were obtained by PCR of the target sequences containing only the DNA parts necessary for Cell-Free Transcription System.Here it is shown only the gel electrophoresis of the T7-HRE-sB-T.

Figure 2:PCR validation of the cell free biosensor template(T7-HRE-sB-T)..


Test and Optimization

To test the created parts, we performed two iterations. Firstly, we tested and optimized the fluorescence output without the presence of any receptor or ligand (Test & Learn I), to ensure that the design at its basic level works properly. Secondly, we proceeded by testing the biosensor on its whole with the receptor and the ligands (Test & Learn II).

1. Wavelength and Plate reader setting

  • Rationale: As the signal for the first experiments was erratic and sometimes incoherent, we tried to improve the reading settings.
  • Result:Higher fluorescence output was yielded by: 
Using the wavelength couple 488/530 nm. 
Reading from the top (instead from the bottom)

Figure 5: Wavelength optimization.Fluorescein sodium salt was used as reference.
2. Buffer test

  • Rationale: Optimize the reaction to increase the signal

Initially, for the first transcription test, we used a custom In Vitro Transcription (IVT) buffer recommended from the ROSALIND protocol (where we took the inspiration for the cell-free system). However, we didn’t get any fluorescence emission by using that custom buffer.

  • Result: The commercial In Vitro Transcription (IVT) buffer was better than the custom made.

When using the commercial buffer, we could see a much higher output signal. The custom buffer clearly is not ideal for the cell-free transcription, while the commercial buffer seems to work much better. A possible explanation for this, is the ionic concentration, which is much higher in the custom buffer (especially NaCl). Either the indicated concentrations were wrong (we in fact acknowledge a mistake in the ROSALIND protocol, as the indicated concentration of the NaCl ion was too high) or we made a mistake in the process of preparing it.

3. DNA concentration

  • Rationale: Increase the sensitivity of the biosensor, enlarge the limits of detection, and reduce the cost of testing.

Reducing the amount of DNA enhances both sensitivity and the limit of detection, which are critical factors for our stakeholders. This reduction will ultimately lower the detection threshold, allowing even trace amounts of EDCs in the tested water to produce measurable inhibition. Additionally, it enables us to minimize the use of the receptor, the most expensive component of the biosensor system.

  • Results: A concentration of 10 nM of DNA was sufficient to yield a substantial signal and the best one to perform the next experiments, also according to the Modeling analysis.

Figure 6: Test for different DNA concentrations in order to have a readable and measurable signal. Measurements were taken after 1 hour for 20 minutes. Error bars represent standard deviation of triplicates.


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]



References

  1. Chen, R., Cheng, H., Jin, P., Song, L., Yue, T., Hull, M., & Mansell, T. J. (2020). Nature Biotechnology, 38(10), 1107–1112. https://doi.org/10.1038/s41587-020-0571-7