Difference between revisions of "Part:BBa K5477051"

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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.

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.

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.

</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.

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.