Team:Freiburg/Improve

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Overview Motivation CAR T Cells Tumor Microenvironment AND Gate Outlook Achievements References
Members Attributions Partners Contact
Main Project Modeling Applied Design Proof of Concept BioBricks Basic Part BioBrick Improvement Interlab Study
Safety Cloning HIF1A Knockdown Cell Culture Lab Book
Human Practice Integrated Human Practice Collaborations

BioBrick Improvement

At the beginning of our project, the iGEM part library was checked for parts that could be used for the CARTELTM AND gate. Since oxygen concentration plays a key role in the tumor microenvironment, we were especially interested in parts containing HREs (Hypoxia Response Elements).
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). We decided to improve BBa_K1720002 (iGEM15_SCUT-China) and BBa_K1456004 (iGEM14_ATOMS-Turkiye).

Since BBa_K1720002 (iGEM15_SCUT-China) contains a CMV promoter, sensitive oxygen dependent gene expression is not possible due to a high basal expression downstream of the CMV promoter. 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).

Cell Culture

Figure 1: BBa_2295003 and multiple enhancer elements.

BBa_K1456004 doesn’t contain a promoter at all. Hence it is not ready to use and a lot of further cloning is required, before oxygen dependent gene expression can be studied. The introduction of multiple enhancer elements allows highly specific oxygen dependent expression in different cell types. Tuning the amount of enhancer elements, downstream transcription can be regulated. By introducing a BglII restriction site between the HRE and the pTal promoter in BBa_K2295003, we offer an easy cloning strategy to change the amount of multiple enhancer elements. Using compatible end cloning, future iGEM teams will profit a lot from the improvement of BBa_K1720002 and BBa_K1456004.