New parts have been added to the registry in order to help teams build more circuits and gain more knowledge about essential parts in our platform. As some parts were improved and descriptive provisions were added through literature and experimental characterization. iGEM teams will have access to a large library of parts provided by our project demonstrated as follows:

We added a new set of 16 parts to the library that vary between basic and composite parts. The new basic parts are:

BBa_K4140004, BBa_K4140010, BBa_K4140013, BBa_K4140014, BBa_K4140015, BBa_K4140018, BBa_K4140019, BBa_K4140020, BBa_K4140021 , BBa_K4140022.

while the composite parts are as follows:

BBa_K4140023, BBa_K4140024, BBa_K4140025, BBa_K4140026, BBa_K4140027, BBa_K4140028.

Future iGEM Teams will be able to build advanced systems and circuits with our new improved parts. Incorporating a peptide signal (KP-SP tagging) into the LacZ alpha gene was our first improvement, as it mediates the extracellular secretion of B-galactosidase (KP-SP tagging) in order to improve the cleavage capacity of the X gal, thus increasing the intensity of the dark blue color emitted ( BBa_K4140008 ). Furthermore, We ultimately optimized the design of the previously mentioned Sensing system in order to limit its off-targeting effect, where we added a kink turn upstream of Cas12g-Coding sequence (L7Ae-Kt-dependant translational regulation of Cas12g) to limit the circuits functionality in the absence of the triggering biomarker, which in our case was phenylalanine. This system that we provided is extremely modular and versatile, as it can be applied in many other diagnostic studies and projects, resulting in better and well-controlled biosensors, of the least potential to explicit false or inaccurate results. The L7Ae-boxC/D k-turn complex inhibits ribosomal function on Cas12g mRNA, acting as a translation off-switch by repressing Cas12g synthesis in the presence of L7Ae.

BBa_K4140016

In order to characterize and model our parts, mathematical modeling and literature characterization have been carried out for multiple parts. Such as

BBa_K4140001, BBa_K4140009, BBa_K4140005, BBa_K4140006, BBa_K4140017, BBa_K4140000, BBa_K4140002, parts page

And through directed evolution, we were able to provide future teams with mutational landscapes demonstrating positive fit mutants, thus enhancing various parts for the ultimate effective performance.

And through directed evolution, we were able to provide future teams with mutational landscapes demonstrating positive fit mutants, thus enhancing various parts for the ultimate effective performance.

BBa_K4140003

And for all the teams who will use the CRISPR technology in the future, we introduced an extra-safety switch through natural brake and regulatory elements for the Crispr system by using anti-crispr proteins. In this regard, we designed an additional regulatory and tuning system for the Crispr itself using anti-crispr proteins that can effectively bind to the CRISPR-Cas system at many different sites, each inhibiting a certain function of the CRISPR.

We also modified this system to be exogenously triggered by tetracycline, releasing anti-crispr proteins capable of counteracting the cleavage mechanism and stopping the crispr in case the circuit gets out of control. A Tet-On 3G inducible system was introduced to the anti-crispr part which works as a modified technique for gene expression control of both the Cre-recombinase and the anti-crispr protein.

In order for Tet-On 3G to activate the TRE3G promoter, an external element (Tet and tet derivatives) such as doxycycline hydrochloride (Dox) must be present, which improves the DNA-binding capacity of Tet-On 3G, performing transcription activation of the TRE3G promoter and expressing our transgene (Cre recombinase) freely, targeting the loxP sites flanking the (STOP) sequence that it’s upstream of the anti-crispr protein. A nuclear localization signal is attached to nLacZ in this stop sequence as a reporter and indicator of the proper function of the circuit. Using this system, the downstream gene cannot be expressed (anti CRISPR). After the deletion of the (STOP) sequence by Cre recombinase activity, the transcription of nLacZ will be replaced by antiCRISPR expression. Thus, we ensure a versatile, safe design that is modular, adaptive, safe and self-regulated system.