Part:BBa_K3284001:Design
TGFbRI/Taz Chimeric Receptor Device
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
In this project, CEAker is a synthetically combined receptor used as the diagnostic tool system. TGFBR1, which is originally found in human body, directly interacts with Carcinoembryonic Antigen (CEA). CEA itself functions as an intercellular adhesion molecule and is upregulated in a wide variety of human cancers[1], including lung cancer. CEA-binding domain mainly lodged in TGFBR1’s extracellular environment. Hence, the domain, boarden between 1th-126th amino acid, was chosen from TGFBR1. Furthermore, Transcriptional Coactivator with PDZ-binding motif (TAZ) consists of Tar receptor, Tar HAMP domain, and EnvZ histidine kinase domain. It presents as an oncogene that is used for the early detection of lung cancer[2]. Therefore, our team selected Taz domains involving 1st-40th and 167th-484th amino acid as part of CEAKEER.
Biobrick presence inside E. coli shows that PCR colony is essential for multiplications of the whole gene. PCR colony uses either specific primers, i.e. forward primer and reverse primer. Forward primer is used for elongation of the ribbon from the front or upper chain, while the reverse primer is the opposite. It is utilized from behind the elongation or bottom chain, for that reason it would produce completed double strands. PCR colony primer can integrate or connect between one fragment to another fragment of our part. CEAKEER PCR colony primers is constructed by using NCBI primer BLAST website. The sequences are served as follows.
Figure 2. Detailed Data for CEAKEER PCR Colony Primers
There are many necessities used to choose the primer. The primers need to be formulated so that they are complementary to a unique sequence of nucleotides “upstream” and “downstream of the sequence of interest”. They can not match a sequence within the area of interest. It would cause PCR to start off too late and miss a portion of the area we want to amplify. Beside, they should also not have complementary regions within themselves, therefore it will fold over and bind themselves, forming a “hairpin”. The forward and reverse primers that is explained above, should not be complementary, or they will anneal to each other and form a “primer dimer”. However, those problems primers can be avoided by primer of 15-20 nucleotides in length or approximately 20 basepairs (bp)[1]. This 20 basepairs classified as small DNA primers and thus the temperature at which the DNA primers anneal to the target DNA sequence depends on the length of the primers and the sequence targeted[2]. On the other hand, the primer temperature has to be around its melting points so it will not occurs denaturation. The standard of melting point of the primer i.e. min. 56-60℃. The score of GC content must be at the range 50-55%, if the GC content is below this range then the primer will not be strongly attached to the DNA mold. Because of the lenght of the primer, the primer can stick with reverse so that it will form a complex bond or join the primer itself, which results in the PCR is going to fail and not being able to work. Proper primary selection is the one of the keys to the success of the process.
Source
The extracellular (CEA-sensing) domain of TGFbRI is a native in Homo sapiens, while the ransmembrane+intracellular domain of Tar/EnvZ receptor is a part from BBa_C0082 Escherichia coli.
References
[1] Di.uq.edu.au. (2017). Polymerase Chain Reaction (PCR). [online]. Available at: https://di.uq.edu.au/community-and-alumni/sparq-ed/sparq-ed-services/polymerase-chain-reaction-pcr#.[Accessed 13 October. 2019].
[2] Peaper, D. R. & Landry, M. L. (2014). Laboratory Diagnosis of Infection. Handbook of Clinical Neurology, 123, 123-147. doi: 10.1016/B978-0-444-53488-0.00005-5.