At the beginning of our project, the iGEM part library was checked for parts that could be used for the CARTEL^TM AND gate. Since oxygen concentration plays a key role in the tumor microenvironment, we were especially interested in parts containing HREs (Hypoxia Response Elements). Though, since those BioBricks only covered the needs of our project partially, we decided to improve BBa_K1720002 (iGEM15_SCUT-China) and BBa_K1456004 (iGEM14_ATOMS-Turkiye). So we sent in BBa_K2295003, to fulfill the needs further iGEM teams, dealing with hypoxia dependent gene expression, might have.

Currently there is no BioBrick containing a minimal promoter in addition to the HRE, but only HRE itself (BBa_K1456004) as well as a combination of CMV-HRE-TATAbox (BBa K1720002).

Since BBa K1720002 (iGEM15_SCUT-China)contains a CMV promoter, sensitive oxygen dependent gene expression is not possible. Having a high basal expression downstream of the CMV promoter, BBa K1720002 is not feasible for many projects. This problem is fixed in our improvement BBa_K2295003 by introducing the minimal promoter pTal downstream of the HRE, instead of a CMV promoter upstream of the HRE. However, to remove an EcoRI restriction side, the pTal promoter was slightly modified by changing one base pair (detailed description of BBa_K2295003).