Difference between revisions of "Part:BBa K5477051"
 
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<h2>Devices</h2>
 
<h2>Devices</h2>
  
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The Devices Table outlines the different biosensor devices developed for the detection and detoxification of various environmental contaminants. Each device incorporates specific receptor modules and reporter modules, engineered to detect particular compounds like polycyclic aromatic hydrocarbons (PAHs), dioxins, polychlorinated biphenyls (PCBs), and bisphenol A (BPA). For instance, devices BBa_K5477041 and BBa_K5477042 are biosensors designed to detect PAHs, dioxins, or dioxin-like PCBs, while devices BBa_K5477043 to BBa_K5477045 are specialized for BPA detection. Additionally, BBa_K5477046 introduces the SUPERMOM device, which integrates both detection and detoxification modules to simultaneously identify and neutralize BPA in breast milk. The final device, BBa_K5477047, is a detoxification device targeting dioxins and PCBs through the use of CYP1A1 and UGT enzymes, which metabolize these contaminants into safer, water-soluble compounds. BBa_K5477048 device was used to test the SUPERMOM concept. This array of devices demonstrates the versatility of the biosensor platform.
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The Devices Table outlines the different biosensor devices developed for the detection and detoxification of various environmental contaminants. Each device incorporates specific receptor modules and reporter modules, engineered to detect particular compounds like polycyclic aromatic hydrocarbons (PAHs), dioxins, polychlorinated biphenyls (PCBs), and bisphenol A (BPA). For instance, devices BBa_K5477041 and BBa_K5477042 are biosensors designed to detect PAHs, dioxins, or dioxin-like PCBs, while devices BBa_K5477043 to BBa_K5477045 are specialized for BPA detection. Additionally, BBa_K5477046 introduces the SUPERMOM device, which integrates both detection and detoxification modules to simultaneously identify and neutralize BPA in breast milk. The final device, BBa_K5477047, is a detoxification device targeting dioxins and PCBs through the use of CYP1A1 and UGT enzymes, which metabolize these contaminants into safer, water-soluble compounds. BBa_K5477048 device was used to test the SUPERMOM concept. This array of devices demonstrates the versatility of the biosensor platform. The results for each part can be accessed through their specific registry page.
  
  
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<h2>Composites</h2>
 
<h2>Composites</h2>
  
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The Composites Table shows the various composite parts that make up the functional modules of each biosensor device. Each composite part consists of a combination of promoters, receptors, or detoxification modules designed for specific detection or detoxification tasks. For example, BBa_K5477023 and BBa_K5477024 feature receptor modules engineered to detect PAHs, dioxins, and dioxin-like PCBs, while BBa_K5477027 and BBa_K5477028 are optimized for BPA detection through engineered estrogen receptor modules. Additionally, the composite parts BBa_K5477035 to BBa_K5477039 focus on detoxification, containing enzymes such as UGT2B15, UGT1A1, and CYP1A1 that metabolize harmful chemicals into less toxic or more easily excretable forms. These composite parts serve as the BioBricks of the devices, providing the necessary functionality to detect and detoxify environmental contaminants.
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The Composites Table shows the various composite parts that make up the functional modules of each biosensor device. Each composite part consists of a combination of promoters, receptors, or detoxification modules designed for specific detection or detoxification tasks. For example, BBa_K5477023 and BBa_K5477024 feature receptor modules engineered to detect PAHs, dioxins, and dioxin-like PCBs, while BBa_K5477027 and BBa_K5477028 are optimized for BPA detection through engineered estrogen receptor modules. Additionally, the composite parts BBa_K5477035 to BBa_K5477039 focus on detoxification, containing enzymes such as UGT2B15, UGT1A1, and CYP1A1 that metabolize harmful chemicals into less toxic or more easily excretable forms. These composite parts serve as the BioBricks of the devices, providing the necessary functionality to detect and detoxify environmental contaminants. To build the composites, USER cloning method was used. For more details about the method, see Parts below.
  
 
<table>
 
<table>
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             <th>Part</th>
 
             <th>Part</th>
 
             <th>Type</th>
 
             <th>Type</th>
 +
            <th>Function</th>
 
             <th>Length (bp)</th>
 
             <th>Length (bp)</th>
 
         </tr>
 
         </tr>
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             <td>pRET2-ARNT receptor module</td>
 
             <td>pRET2-ARNT receptor module</td>
 
             <td>Composite</td>
 
             <td>Composite</td>
−
             <td>Promoter and nuclear translocator detection of PAHs, dioxin or dioxin-like PCBs</td>
+
             <td>Promoter and nuclear translocator for detection of PAHs, dioxin or dioxin-like PCBs</td>
 
             <td>2155</td>
 
             <td>2155</td>
 
         </tr>
 
         </tr>
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             <td>pRET2-NCOA - receptor module</td>
 
             <td>pRET2-NCOA - receptor module</td>
 
             <td>Composite </td>
 
             <td>Composite </td>
−
             <td>Promoter and nuclear receptor co-activator for the detection of
+
             <td>Promoter and nuclear receptor coactivator for the detection of
 
PAHs, dioxin or dioxin-like PCBs</td>
 
PAHs, dioxin or dioxin-like PCBs</td>
 
             <td>5032</td>
 
             <td>5032</td>
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             <td>CYP3A4-MYC-pPDC1-Lex6Op-pENO1-POR</td>
 
             <td>CYP3A4-MYC-pPDC1-Lex6Op-pENO1-POR</td>
 
             <td>Composite</td>
 
             <td>Composite</td>
−
             <td>Detoxification module for BPA</td>
+
             <td>Detoxification module</td>
 
             <td>4193</td>
 
             <td>4193</td>
 
         </tr>
 
         </tr>
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</table>
 
</table>
  
 +
 +
<h2>Cloning Parts into YCp Plasmids</h2>
 +
USER cloning was performed to assemble the parts into YCp plasmids. Different selection markers were utilized for identification, depending on the combinations being constructed. The pUUS vector, optimized for the assembly of reporter plasmids, was used in this process. This approach allowed for flexibility in building various combinations of biosensor components.
 +
 +
 +
<h2>Transformation of and Validation </h2>
 +
Once assembled, the plasmids were first transformed into <i>E. coli</i> for propagation and then into yeast for functional testing. The success of the cloning and transformation was evaluated through gel electrophoresis and sequencing to confirm the correct constructs. The yeast was engineered to contain biosensor devices with specific combinations of receptor and reporter modules. The design of complementary overhangs in the USER-ready parts allowed for flexibility in assembling different parts, enabling the combinations of various composite constructs engineered to specific functionalities. In some cases, a detoxification function was incorporated, resulting in engineered yeast capable of both detecting and detoxifying contaminants. This engineered yeast, named SUPERMOM, aims to detect and detoxify environmental contaminants. The following gels below show the constructs built with the USER-ready parts and YCp backbones. The ladder is 1 Kb Plus DNA ladder.
 +
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<html><div style="text-align: center;"><img src="https://static.igem.wiki/teams/5477/for-registry/gels/gel1-comp.webp" width="500"></div></html>
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<html><div style="text-align: center;"><img src="https://static.igem.wiki/teams/5477/for-registry/gels/gel2-comp.webp" width="500"></div></html>
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<html><div style="text-align: center;"><img src="https://static.igem.wiki/teams/5477/for-registry/gels/gel3-comp.webp" width="400"></div></html>
  
  
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<h2>Parts</h2>
 
<h2>Parts</h2>
  
−
The Parts Table lists individual genetic components, including promoters, coding sequences (CDS), and regulatory elements, that make up the biosensor and detoxification systems. Promoters such as pRET2 and pSTE12 are used to drive constitutive or inducible expression of the receptor and detoxification modules. The table also includes various coding sequences for receptors like AhR, ARNT, and LexA-ERα, which are central to detecting endocrine disruptors like BPA and other toxic chemicals. Detoxification enzymes such as CYP1A1, UGT2B15, and CYP3A4 are also featured, responsible for metabolizing and neutralizing harmful substances. These individual parts or BioBricks allow the biosensors to be modular, enabling customization for different contaminants while maintaining compatibility with standard assembly method RFC1000.
+
The Parts Table lists individual genetic components, including promoters, coding sequences (CDS), and regulatory elements, that make up the biosensor and detoxification systems. Promoters such as pRET2 and pSTE12 are used to drive constitutive or inducible expression of the receptor and detoxification modules. The table also includes various coding sequences for receptors like AhR, ARNT, and LexA-ERα, which are central to detecting endocrine disruptors like BPA and other toxic chemicals. Detoxification enzymes such as CYP1A1, UGT2B15, and CYP3A4 are also featured, responsible for metabolizing and neutralizing harmful substances. These individual parts or BioBricks allow the biosensors to be modular, enabling customization for different contaminants while maintaining compatibility with standard assembly method RFC1000.  
  
  
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             <th>Type</th>
 
             <th>Type</th>
 
             <th>Length (bp)</th>
 
             <th>Length (bp)</th>
−
         </tr>
+
         </tr>  
 
         <tr>
 
         <tr>
 
             <td> [https://parts.igem.org/Part:BBa_K5477000 BBa_K5477000]</td>
 
             <td> [https://parts.igem.org/Part:BBa_K5477000 BBa_K5477000]</td>
−
             <td>pRET2 - Medium strong constitutive promoter in Saccharomyces cerevisiae</td>
+
             <td>pRET2 - Medium strong constitutive promoter in <i>Saccharomyces cerevisiae</i></td>
 
             <td>Regulatory (Promoter)</td>
 
             <td>Regulatory (Promoter)</td>
 
             <td>700</td>
 
             <td>700</td>
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         <tr>
 
         <tr>
 
             <td>[https://parts.igem.org/Part:BBa_K5477001 BBa_K5477001]</td>
 
             <td>[https://parts.igem.org/Part:BBa_K5477001 BBa_K5477001]</td>
−
             <td>pSTE12 - Constitutive promoter in Saccharomyces cerevisiae</td>
+
             <td>pSTE12 - Constitutive promoter in <i>Saccharomyces cerevisiae</i></td>
 
             <td>Regulatory (Promoter)</td>
 
             <td>Regulatory (Promoter)</td>
 
             <td>557</td>
 
             <td>557</td>
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         <tr>
 
         <tr>
 
             <td>[https://parts.igem.org/Part:BBa_K5477002 BBa_K5477002]</td>
 
             <td>[https://parts.igem.org/Part:BBa_K5477002 BBa_K5477002]</td>
−
             <td>pRAD27 - Weak constitutive promoter in Saccharomyces cerevisiae</td>
+
             <td>pRAD27 - Weak constitutive promoter in <i>Saccharomyces cerevisiae</i></td>
 
             <td>Regulatory (Promoter)</td>
 
             <td>Regulatory (Promoter)</td>
 
             <td>694</td>
 
             <td>694</td>
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         <tr>
 
         <tr>
 
             <td>[https://parts.igem.org/Part:BBa_K5477003 BBa_K5477003]</td>
 
             <td>[https://parts.igem.org/Part:BBa_K5477003 BBa_K5477003]</td>
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             <td>pMEL1 - Constitutive core promoter in Saccharomyces cerevisiae</td>
+
             <td>pMEL1 - Constitutive core promoter in <i>Saccharomyces cerevisiae</i></td>
 
             <td>Regulatory (Promoter)</td>
 
             <td>Regulatory (Promoter)</td>
 
             <td>256</td>
 
             <td>256</td>
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         <tr>
 
         <tr>
 
             <td>[https://parts.igem.org/Part:BBa_K5477004 BBa_K5477004]</td>
 
             <td>[https://parts.igem.org/Part:BBa_K5477004 BBa_K5477004]</td>
−
             <td>pLEU2 - Constitutive core promoter in Saccharomyces cerevisiae</td>
+
             <td>pLEU2 - Constitutive core promoter in <i>Saccharomyces cerevisiae</i></td>
 
             <td>Regulatory (Promoter)</td>
 
             <td>Regulatory (Promoter)</td>
 
             <td>130</td>
 
             <td>130</td>
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         <tr>
 
         <tr>
 
             <td>[https://parts.igem.org/Part:BBa_K5477005 BBa_K5477005]</td>
 
             <td>[https://parts.igem.org/Part:BBa_K5477005 BBa_K5477005]</td>
−
             <td>pGAL1/10 - Bidirectional inducible promoters from Saccharomyces cerevisiae</td>
+
             <td>pGAL1/10 - Bidirectional inducible promoters from <i>Saccharomyces cerevisiae</i></td>
 
             <td>Regulatory (Promoter)</td>
 
             <td>Regulatory (Promoter)</td>
 
             <td>664</td>
 
             <td>664</td>
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         <tr>
 
         <tr>
 
             <td>[https://parts.igem.org/Part:BBa_K5477007 BBa_K5477007]</td>
 
             <td>[https://parts.igem.org/Part:BBa_K5477007 BBa_K5477007]</td>
−
             <td>pPOP6 - Weak constitutive promoter in Saccharomyces cerevisiae</td>
+
             <td>pPOP6 - Weak constitutive promoter in <i>Saccharomyces cerevisiae</i></td>
 
             <td>Regulatory (Promoter)</td>
 
             <td>Regulatory (Promoter)</td>
 
             <td>700</td>
 
             <td>700</td>
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             <td>[https://parts.igem.org/Part:BBa_K5477008 BBa_K5477008]</td>
 
             <td>[https://parts.igem.org/Part:BBa_K5477008 BBa_K5477008]</td>
 
             <td>XRE - Xenobiotic Response Element </td>
 
             <td>XRE - Xenobiotic Response Element </td>
−
             <td>Regulatory Binding Element </td>
+
             <td>Regulatory - Binding Element </td>
 
             <td>82</td>
 
             <td>82</td>
 
         </tr>
 
         </tr>
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             <td>[https://parts.igem.org/Part:BBa_K5477009 BBa_K5477009]</td>
 
             <td>[https://parts.igem.org/Part:BBa_K5477009 BBa_K5477009]</td>
 
             <td>Lex6Op - six tandem copies of LexA operator</td>
 
             <td>Lex6Op - six tandem copies of LexA operator</td>
−
             <td>Regulatory Binding Element </td>
+
             <td>Regulatory - Binding Element </td>
 
             <td>220</td>
 
             <td>220</td>
 
         </tr>
 
         </tr>
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             <td>[https://parts.igem.org/Part:BBa_K3793005 BBa_K3793005]</td>
 
             <td>[https://parts.igem.org/Part:BBa_K3793005 BBa_K3793005]</td>
 
             <td>ARNT - Aryl hydrocarbon Nuclear Transporter</td>
 
             <td>ARNT - Aryl hydrocarbon Nuclear Transporter</td>
−
             <td>CDS - receptor </td>
+
             <td>CDS - coactivator </td>
 
             <td>2370</td>
 
             <td>2370</td>
 
         </tr>
 
         </tr>
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             <td>[https://parts.igem.org/Part:BBa_K5477012 BBa_K5477012]</td>
 
             <td>[https://parts.igem.org/Part:BBa_K5477012 BBa_K5477012]</td>
 
             <td>NCOA - Nuclear receptor coactivator </td>
 
             <td>NCOA - Nuclear receptor coactivator </td>
−
             <td>CDS - receptor</td>
+
             <td>CDS - coactivator</td>
 
             <td>4326</td>
 
             <td>4326</td>
 
         </tr>
 
         </tr>
 
         <tr>
 
         <tr>
 
             <td>[https://parts.igem.org/Part:BBa_K5477013 BBa_K5477013]</td>
 
             <td>[https://parts.igem.org/Part:BBa_K5477013 BBa_K5477013]</td>
−
             <td>LexA-ERα chimeric activator</td>
+
             <td>LexA-ERα(LBD) chimeric activator</td>
 
             <td>CDS - receptor</td>
 
             <td>CDS - receptor</td>
 
             <td>1683</td>
 
             <td>1683</td>
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         <tr>
 
         <tr>
 
             <td>[https://parts.igem.org/Part:BBa_K5477014 BBa_K5477014]</td>
 
             <td>[https://parts.igem.org/Part:BBa_K5477014 BBa_K5477014]</td>
−
             <td>LexA-mERα chimeric activator</td>
+
             <td>LexA-mERα(LBD) chimeric activator</td>
 
             <td>CDS - receptor</td>
 
             <td>CDS - receptor</td>
 
             <td>1683</td>
 
             <td>1683</td>
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         <tr>
 
         <tr>
 
             <td>[https://parts.igem.org/Part:BBa_K5477015 BBa_K5477015]</td>
 
             <td>[https://parts.igem.org/Part:BBa_K5477015 BBa_K5477015]</td>
−
             <td>LexA-ERRγ chimeric activator</td>
+
             <td>LexA-ERRγ(LBD) chimeric activator</td>
 
             <td>CDS - receptor</td>
 
             <td>CDS - receptor</td>
 
             <td>1602</td>
 
             <td>1602</td>
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     </table>
 
     </table>
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<h2>USER Cloning Overview</h2>
 +
 +
Uracil-Specific Excision Reaction (USER) cloning is a ligase-independent technique that facilitates DNA fragment assembly by creating specific overhangs through the incorporation of uracil in primers (1). The process uses Q5U Hot Start DNA polymerase to introduce uracil into the PCR amplicons. After digestion with Uracil DNA Glycosylase (UDG) and Endonuclease VIII, these overhangs allow fragments to hybridize directly with the vector, enabling rapid and efficient cloning without the need for ligase. This method is particularly useful for assembling multiple fragments into a single construct.
 +
 +
 +
<h3> Preparing USER-ready Parts </h3>
 +
Primers were designed to incorporate uracil for specific overhangs to make the desired parts USER-ready. In cases where the genes were too large, they were ordered in smaller segments, and additional primers were designed to create fragments compatible with USER cloning. This can be seen for instance on parts occurring twice like AhR. This approach ensured that all components could be efficiently assembled using the USER method. Below are the gels for the parts where the ladder is 1 Kb Plus DNA ladder.
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<html><div style="text-align: center;"><img src="https://static.igem.wiki/teams/5477/for-registry/gels/gel1-parts.webp" width="500"></div></html>
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<html><div style="text-align: center;"><img src="https://static.igem.wiki/teams/5477/for-registry/gels/gel2-parts.webp" width="500"></div></html>
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 +
===References===
 +
 +
1. Geu-Flores F, Nour-Eldin HH, Nielsen MT, Halkier BA. USER fusion: a rapid and efficient method for simultaneous fusion and cloning of multiple PCR products. Nucleic Acids Res. 2007;35(7):e55. doi: 10.1093/nar/gkm106. Epub 2007 Mar 27. PMID: 17389646; PMCID: PMC1874642.

Latest revision as of 12:48, 2 October 2024


Team UCopenhagen 2024: Parts Collection of MilkClear

Introduction

Our part collection includes 48 total parts: 24 basic parts, 16 composite parts, and 8 devices, of which 45 are new. This collection is coherent, as all the parts were designed, built and tested with the specific goal to address contaminants in breast milk. It is also the first collection tackling this issue.

The parts can be categorized into three main categories:

  1. Parts for detecting contaminants in breast milk
  2. Parts for detoxifying contaminants
  3. Devices integrating these functions

All parts in this collection have been tested in either the Wet-lab or the Dry-lab, which demonstrates their functionality. Additionally, we cloned constructs that are not included in this part collection. By contributing a comprehensive collection of receptor, reporter, and detoxification modules, we provide valuable resources that expand current biosensing capabilities and empower future iGEM teams to develop more sophisticated, multi-functional systems. Instead of function, we have chosen complexity to group our parts on this page, as multiple parts reappear in each category due to the interlinked nature of our collection.


Devices

The Devices Table outlines the different biosensor devices developed for the detection and detoxification of various environmental contaminants. Each device incorporates specific receptor modules and reporter modules, engineered to detect particular compounds like polycyclic aromatic hydrocarbons (PAHs), dioxins, polychlorinated biphenyls (PCBs), and bisphenol A (BPA). For instance, devices BBa_K5477041 and BBa_K5477042 are biosensors designed to detect PAHs, dioxins, or dioxin-like PCBs, while devices BBa_K5477043 to BBa_K5477045 are specialized for BPA detection. Additionally, BBa_K5477046 introduces the SUPERMOM device, which integrates both detection and detoxification modules to simultaneously identify and neutralize BPA in breast milk. The final device, BBa_K5477047, is a detoxification device targeting dioxins and PCBs through the use of CYP1A1 and UGT enzymes, which metabolize these contaminants into safer, water-soluble compounds. BBa_K5477048 device was used to test the SUPERMOM concept. This array of devices demonstrates the versatility of the biosensor platform. The results for each part can be accessed through their specific registry page.


Part Name Part Type Function
BBa_K5477041 BBa_K5477023: pSTE12-AhR - receptor module, BBa_K5477025: pRET2-ARNT receptor module, BBa_K5477026: pRET2-NCOA - receptor module, BBa_K5477030: XRE-pMEL1-NanoLuc reporter module Device Biosensor device for detection of PAHs, dioxin or dioxin-like PCBs
BBa_K5477042 BBa_K5477024: pRAD27-AhR - receptor module, BBa_K5477025: pRET2-ARNT receptor module, BBa_K5477026: pRET2-NCOA - receptor module, BBa_K5477030: XRE-pMEL1-NanoLuc reporter module Device Biosensor device for detection of PAHs, dioxin or dioxin-like PCBs
BBa_K5477043 BBa_K5477027: pRET2-LexA-ERα(LBD) - receptor module, BBa_K5477031: Lex6Op-pLEU2-NanoLuc reporter module Device Biosensor device for detection of BPA
BBa_K5477044 BBa_K5477028: pPOP6-LexA-ERα(LBD) - receptor module, BBa_K5477031: Lex6Op-pLEU2-NanoLuc reporter module Device Biosensor device for detection of BPA
BBa_K5477045 BBa_K5477029: pRET2-LexA-mERα(LBD) - receptor module, BBa_K5477031: Lex6Op-pLEU2-NanoLuc reporter module Device Biosensor device for detection of BPA
BBa_K5477046 BBa_K5477029: pRET2-LexA-mERα(LBD) - receptor module, BBa_K5477031: Lex6Op-pLEU2-NanoLuc reporter module, BBa_K5477040:UDPD-pPDC1-Lex6Op-pENO1-UGT2B15 Device SUPERMOM: Dual-Function Biosensor for BPA Detection and Detoxification
BBa_K5477047 BBa_K5477037: CYP1A1-pGAL1/10-POR detox module, BBa_K5477036: UDPD-pGAL1/10-UGT1A1 detox module Device Detoxification device against dioxin and PCBs
BBa_K5477048 BBa_K5477029: pRET2-LexA-mERα(LBD) - receptor module, BBa_K5477031: Lex6Op-pLEU2-NanoLuc reporter module, BBa_K5477039: CYP3A4-MYC-pPDC1-Lex6Op-pENO1-POR Device Testing of the SUPERMOM Concept


Composites

The Composites Table shows the various composite parts that make up the functional modules of each biosensor device. Each composite part consists of a combination of promoters, receptors, or detoxification modules designed for specific detection or detoxification tasks. For example, BBa_K5477023 and BBa_K5477024 feature receptor modules engineered to detect PAHs, dioxins, and dioxin-like PCBs, while BBa_K5477027 and BBa_K5477028 are optimized for BPA detection through engineered estrogen receptor modules. Additionally, the composite parts BBa_K5477035 to BBa_K5477039 focus on detoxification, containing enzymes such as UGT2B15, UGT1A1, and CYP1A1 that metabolize harmful chemicals into less toxic or more easily excretable forms. These composite parts serve as the BioBricks of the devices, providing the necessary functionality to detect and detoxify environmental contaminants. To build the composites, USER cloning method was used. For more details about the method, see Parts below.

Part Name Part Type Function Length (bp)
BBa_K5477023 pSTE12-AhR - receptor module Composite Promoter and Receptor for detection of PAHs, dioxin or dioxin-like PCBs 3117
BBa_K5477024 pRAD27-AhR - receptor module Composite Promoter and Receptor for detection of PAHs, dioxin or dioxin-like PCBs 1900
BBa_K5477025 pRET2-ARNT receptor module Composite Promoter and nuclear translocator for detection of PAHs, dioxin or dioxin-like PCBs 2155
BBa_K5477026 pRET2-NCOA - receptor module Composite Promoter and nuclear receptor coactivator for the detection of PAHs, dioxin or dioxin-like PCBs 5032
BBa_K5477027 pRET2-LexA-ERα(LBD) - receptor module Composite Promoter and Receptor for detection of BPA 2389
BBa_K5477028 pPOP6-LexA-ERα(LBD) - receptor module Composite Promoter and Receptor for detection of BPA 2389
BBa_K5477029 pRET2-LexA-mERα(LBD) - receptor module Composite Promoter and Receptor for detection of BPA 2389
BBa_K5477030 XRE-pMEL1-NanoLuc - reporter module Composite A xenobiotic response element, a promoter, and a luciferase reporter gene for the detection signal against PCBs, dioxins, and dioxin-like compounds 868
BBa_K5477031 Lex6Op-pLEU2-NanoLuc reporter module Composite DNA-binding domain, promoter, and luciferase reporter gene for the detection signal against BPA 880
BBa_K5477035 UDPD-pGAL1/10-UGT2B15 Composite Detoxification module against BPA 3724
BBa_K5477036 UDPD-pGAL1/10-UGT1A1 Composite Detoxification module against dioxin and PCBs 3726
BBa_K5477037 CYP1A1-pGAL1/10-POR Composite Detoxification module against dioxin and PCBs 4312
BBa_K5477038 CYP3A4-pGAL1/10-POR Composite Detoxification module against a wide array of contaminants 4250
BBa_K5477039 CYP3A4-MYC-pGAL1/10-POR Composite Detoxification module against a wide array of contaminants 4286
BBa_K5477040 UDPD-pPDC1-Lex6Op-pENO1-UGT2B15 Composite Detoxification module for the detoxification of BPA 3631
BBa_K5477032 CYP3A4-MYC-pPDC1-Lex6Op-pENO1-POR Composite Detoxification module 4193


Cloning Parts into YCp Plasmids

USER cloning was performed to assemble the parts into YCp plasmids. Different selection markers were utilized for identification, depending on the combinations being constructed. The pUUS vector, optimized for the assembly of reporter plasmids, was used in this process. This approach allowed for flexibility in building various combinations of biosensor components.


Transformation of and Validation

Once assembled, the plasmids were first transformed into E. coli for propagation and then into yeast for functional testing. The success of the cloning and transformation was evaluated through gel electrophoresis and sequencing to confirm the correct constructs. The yeast was engineered to contain biosensor devices with specific combinations of receptor and reporter modules. The design of complementary overhangs in the USER-ready parts allowed for flexibility in assembling different parts, enabling the combinations of various composite constructs engineered to specific functionalities. In some cases, a detoxification function was incorporated, resulting in engineered yeast capable of both detecting and detoxifying contaminants. This engineered yeast, named SUPERMOM, aims to detect and detoxify environmental contaminants. The following gels below show the constructs built with the USER-ready parts and YCp backbones. The ladder is 1 Kb Plus DNA ladder.



Parts

The Parts Table lists individual genetic components, including promoters, coding sequences (CDS), and regulatory elements, that make up the biosensor and detoxification systems. Promoters such as pRET2 and pSTE12 are used to drive constitutive or inducible expression of the receptor and detoxification modules. The table also includes various coding sequences for receptors like AhR, ARNT, and LexA-ERα, which are central to detecting endocrine disruptors like BPA and other toxic chemicals. Detoxification enzymes such as CYP1A1, UGT2B15, and CYP3A4 are also featured, responsible for metabolizing and neutralizing harmful substances. These individual parts or BioBricks allow the biosensors to be modular, enabling customization for different contaminants while maintaining compatibility with standard assembly method RFC1000.


Part Name Part Type Length (bp)
BBa_K5477000 pRET2 - Medium strong constitutive promoter in Saccharomyces cerevisiae Regulatory (Promoter) 700
BBa_K5477001 pSTE12 - Constitutive promoter in Saccharomyces cerevisiae Regulatory (Promoter) 557
BBa_K5477002 pRAD27 - Weak constitutive promoter in Saccharomyces cerevisiae Regulatory (Promoter) 694
BBa_K5477003 pMEL1 - Constitutive core promoter in Saccharomyces cerevisiae Regulatory (Promoter) 256
BBa_K5477004 pLEU2 - Constitutive core promoter in Saccharomyces cerevisiae Regulatory (Promoter) 130
BBa_K5477005 pGAL1/10 - Bidirectional inducible promoters from Saccharomyces cerevisiae Regulatory (Promoter) 664
BBa_K5477006 pPDC1-Lex6Op-pENO1 - Bidirectional synthetic promoters with Lex6Op Regulatory (Promoter) 571
BBa_K5477007 pPOP6 - Weak constitutive promoter in Saccharomyces cerevisiae Regulatory (Promoter) 700
BBa_K5477008 XRE - Xenobiotic Response Element Regulatory - Binding Element 82
BBa_K5477009 Lex6Op - six tandem copies of LexA operator Regulatory - Binding Element 220
BBa_K3793004 AhR - Aryl hydrocarbon Receptor CDS - receptor 2547
BBa_K3793005 ARNT - Aryl hydrocarbon Nuclear Transporter CDS - coactivator 2370
BBa_K1680009 NanoLuc - NanoLuciferase reporter gene CDS - Reporter gene 516
BBa_K5477012 NCOA - Nuclear receptor coactivator CDS - coactivator 4326
BBa_K5477013 LexA-ERα(LBD) chimeric activator CDS - receptor 1683
BBa_K5477014 LexA-mERα(LBD) chimeric activator CDS - receptor 1683
BBa_K5477015 LexA-ERRγ(LBD) chimeric activator CDS - receptor 1602
BBa_K5477016 UGT2B15 CDS - enzyme 1593
BBa_K5477017 UGT1A1 CDS - enzyme 1602
BBa_K5477018 UDPD CDS - enzyme 1446
BBa_K5477019 CYP1A1 CDS - receptor 1575
BBa_K5477020 CYP3A4 CDS - enzyme 1512
BBa_K5477021 CYP3A4-MYC CDS - enzyme 1548
BBa_K5477022 POR CDS - enzyme 2061

USER Cloning Overview

Uracil-Specific Excision Reaction (USER) cloning is a ligase-independent technique that facilitates DNA fragment assembly by creating specific overhangs through the incorporation of uracil in primers (1). The process uses Q5U Hot Start DNA polymerase to introduce uracil into the PCR amplicons. After digestion with Uracil DNA Glycosylase (UDG) and Endonuclease VIII, these overhangs allow fragments to hybridize directly with the vector, enabling rapid and efficient cloning without the need for ligase. This method is particularly useful for assembling multiple fragments into a single construct.


Preparing USER-ready Parts

Primers were designed to incorporate uracil for specific overhangs to make the desired parts USER-ready. In cases where the genes were too large, they were ordered in smaller segments, and additional primers were designed to create fragments compatible with USER cloning. This can be seen for instance on parts occurring twice like AhR. This approach ensured that all components could be efficiently assembled using the USER method. Below are the gels for the parts where the ladder is 1 Kb Plus DNA ladder.




References

1. Geu-Flores F, Nour-Eldin HH, Nielsen MT, Halkier BA. USER fusion: a rapid and efficient method for simultaneous fusion and cloning of multiple PCR products. Nucleic Acids Res. 2007;35(7):e55. doi: 10.1093/nar/gkm106. Epub 2007 Mar 27. PMID: 17389646; PMCID: PMC1874642.