Team:Evry Paris-Saclay/Best Part

IGEM Evry Paris-Saclay



The Universal Biosensing Chassis,

our Best Composite Biobrick

Principle and Usages

The Universal Biosensing Chassis (UBC) is the keystone of our project. It aims to provide an answer to the lack of rapid and reliable building methods for transcription-factor based biosensors. Biosensors rely on a basic theoretical principle: a certain concentration of a molecule of interest induces a proportional production of a fluorescent compound. Transcription-factor based biosensors allow the precise and cheap detection or quantification of various chemical compounds.

Using our composite biobrick, one will only need a suitable transcription factor, able to bind to the molecule of interest, and its related promoter. However sometimes, there is no transcription factor in the databases that matches what we are looking for. Here we can use indirect sensing or Sensing-Enabling Metabolic Pathways. Essentially, tools like SensiPath search and design the enzymatic reactions necessary to transform your non-detectable molecule in a detectable one, therefore expanding the repertoire of molecules available to sensing.

Apart from its fast building design, the UBC has been engineered to be used in high throughput procedures such as screening. Indeed, we originally designed it for a new enzyme engineering process for D-psicose production, using a psicose biosensor to identify the enzyme mutants showing the best activity. In addition, by its design, the UBC allows fast cloning: depending on what is to be inserted into the UBC and one’s mastery of the Golden Gate Assembly, a functional biosensor can be obtained in less than a week.

Features

Figure 1 : Schematic design of the UBC.
Figure 1. Schematic design of the UBC.


The highly modular architecture of the UBC (Figure 1) allows rapid engineering of your biosensor:

  • Standardized Fusion sites for Golden Gate Assembly: The UBC has been design to facilitate the DNA insertion hence improving speed and ease of construction. You will only need transcription factors and promoters flanked by BsmBI or BbsI, respectively, with appropriate cutting sites for these type IIS restriction enzymes 5’-TGGA and GCAG-3’ (Figures 2 and 3).
  • Many restriction sites: If Golden Gate assembly isn’t convenient for you, we have included various restriction sites to allow the insertion of promoters and transcription factors (with the RBS) using traditional digestion-ligation protocol (Figures 2 and 3).
  • Insertion markers: In order to enable quick and easy identification of the right clones, we put two different reporter genes : mEmerald (BBa_K2448001) for the transcription factors and LacZ-alpha (BBa_K2448003) for the promoters. If markers are an issue for you, a shorter version of the UBC also exists in the registry (BBa_K2448024).
  • An inducible promoter to control the transcription factor expression: pTacI (BBa_K864400). This well-known promoter is IPTG inducible and remains very strong. To better regulate its expression, and consequently the production of the transcription factor, it could be worthwhile to use our pSB1C3 LacIq (BBa_K2448038) as vector.
  • An efficient reporter: mCherry (BBa_K2448004). This fluorescent monomeric protein widely used in biotechnology is derived from the RFP. Its rapid maturation, low brightness as well as its improved photostability and resistance to bleaching makes it the perfect reporter for biosensors for precise measurements. Moreover, unlike GFP like proteins, there is no cell auto-fluorescence effect at its excitation wavelength.
  • Strong RBSs (BBa_K2448000, BBa_K2448002, BBa_K2066527) and efficient synthetic terminators (BBa_B0015, BBa_K2448018).

Usage

The Golden Gate Assembly building process combined with the presence of insertion markers naturally mark the UBC for high throughput screening experiments. It enables the characterization of many parts at once, coupled with a Fluorescence plate reader or Flow Cytometer. Indeed, insertion of various promoters or transcription factors in parallel could not only help in screening banks of promoters, RBS or transcription factors by measuring the fluorescence changes under various conditions, but also banks of enzyme mutants. By comparing the fluorescence, using the same biosensor, the more signal you observe, the more compound of interest there would be.

User Manual

To build a biosensor, you will need to insert a promoter (Figure 3) and its associated transcription factor (Figure 2).

Figure 2. Principle of the insertion of transcription factors in UBC.
Figure 2: Principle of the insertion of transcription factors in UBC.


Figure 3. Principle of the insertion of promoters in UBC.
Figure 3. Principle of the insertion of promoters in UBC.


Inserting the promoter first is recommended for two reasons:

  • • The insertion marker (LacZ-alpha) allows the manipulator to see dark-green or yellow-green colonies (Figure 4) depending on whether the UBC is carrying your promoter (the yellow-green colonies) or not (the dark-green colonies). If the transcription factor is inserted first, the manipulator will have to distinguish blue colonies from dark-green ones, which can be difficult.
  • • Inserting the promoter first, generally a short DNA sequence, allows you to readily see the size variation of a colony PCR product on an agarose gel because the overall construct is still short. To observe a hundred-base pair variation in a 3 or 4 kb construction can represent a greater challenge.

Note: Colonies carrying the UBC with an inserted promoter can range from yellow green to brown on IPTG-X-Gal plates, depending on the inserted promoter strength.

Figure 4. Picture of a plate containing colonies transformed with a Golden Gate reaction mixture. The Golden Gate assembly, performed on the UBC, aimed to replace LacZ-alpha by a promoter. We can clearly see on the picture a majority of yellow-green colonies (with the promoter) and some dark-green ones (with LacZ-alpha).
Figure 4. Picture of a plate containing colonies transformed with a Golden Gate reaction mixture. The Golden Gate assembly, performed on the UBC, aimed to replace LacZ-alpha by a promoter. We can clearly see on the picture a majority of yellow-green colonies (with the promoter) and some dark-green ones (with LacZ-alpha).

To perform the assembly you can, as mentioned above, use the Golden Gate Assembly or traditional digestion-ligation (Figures 2 and 3).

If you use Golden Gate, the UBC uses two different type IIS restriction enzymes, BbsI for the insertion of the promoter and BsmBI for the insertion of the transcription factor. Using these enzymes for this purpose allow the manipulator to use the more common BsaI for some other experiments like, for example, the insertion of the UBC into another construct, with a simple PCR reaction.

In order to insert your transcription factor and promoters, you need to have compatible fusion sites on it, hence 5’-TGGA and GCAG-3’, and a BbsI (for promoters) or BsmBI (for transcription factors) site (Figures 2 and 3). If you use synthesized DNA, you will have to anticipate this. If you didn’t or if you use DNA you didn’t get synthesized yourself, you can easily get fusion and restriction sites with a PCR and the right set of primers.

Part Collection

We constructed based on this UBC 8 different highly specific biosensors: a fructose biosensor (BBa_K2448032) and 7 psicose biosensors (BBa_K2448025, BBa_K2448026, BBa_K2448027, BBa_K2448028, BBa_K2448029, BBa_K2448030 and BBa_K2448031), each of them with a particular dynamic range and sensitivity. This collection could be the first step of a larger repository composed of standardized biosensors for a large number of molecules.

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