Team:UANL Mty-Mexico/Contributions/UANLBricks

In our Project, one of the main contributions has been the generation of a new genetic circuit construction standard, called "UANL Bricks Assembly". This standard has arisen from the need of an alternative way for device construction from pieces containing some of the restrictions sites of the classic biobrick standard, besides having the ability to have more options when performing cuts and ligations of the genetic elements. The structure and origin of our new standard is explained next.

At the time of carrying out the device construction to integrate, the main idea was use the classic biobrick standard. However, it was observed that some of the genetic elements used for this construction had the PstI, EcoRI y SpeI sites, avoiding it from being used.

For these reasons, we opted for the generation of a new standard that will meet our needs. That was why we generated the UANL Bricks, having the following structure:

The main reason why we chose these sites is explained next:

ApaI Site: This site was chosen because of its wide distribution in the polylinkers of many of the most used vectors in molecular biology.

SpeI and XbaI sites: Coming from the Biobrick classic standard, these sites generated compatibles sticky ends which can be used for the union of elements of interest. however, Its important to point that the SpeI site is present in our ccas gene.

XhoI and SalI sites: Due to the complications presented in the ccas gene, it was necessary to introduce a second set of sites for the union of elements, which in this case were XhoI and SalI sites. Further, the SalI site also could be use like PstI was it in the classic standard because it's present en most of the vectors polylinkers.

The use of this second pair of sites allow us have versatility at the moment of the elements union for the generation of devices or circuits in a Biobrick way, increasing the variability of options for constructions.

Therefore, If the parts do not have XbaI or SpeI inside of them, construction could be performed using ApaI and SalI as EcoRi and PstI, respectively. However, if the parts do have Xba or SpeI sites, the constructions could be performed as show in the following scheme.

The next subject to evaluate of our new standard was the viability of itself, reason why we did a search of the economics, practical and useful aspects of the chosen enzymes.

First, we did a search for the prices, concentrations and compatibility of the used enzymes was made, obtaining the first two aspects was equivalent in both standards (classic and UANL bricks), besides they all work at 100% in buffer 4 of New England Biolabs.

Also, we evaluated the capacity of these enzymes to cleavage close to the end of linear DNA fragments, which would be useful when directly cut PCR products.

Figure 2. Simulations of the IPTG induced Biphasic Switch at different IPTG concentrations. In blue is represent the simulations at IPTG 1300 nM; in red, IPTG 1950 nM; in green, IPTG 650 nM; and in cyan, 130 nM.

Finally, we search the distributions of UANL Brick's sites in vectors used in molecular biology, in order to know which ones could be used for cloning. In this study we include companies like Fermentas ®, Promega ®, Invitrogen ®, New England Biolabs ®, besides the classic biobrick vector pSB1A2.