We made a measurement with TOP10 about existing parts BBa_K912000 and BBa_K012001.
We tried to construct four inserts in total for our project. “INSERT1” is for lac I synthesis. It consists of four different biological parts: J23100, B0032, C0012, and B0015. J23100 is a constitutive promoter. B0032 is a ribosome-binding site (RBS). C0012 is a lac I repressor with LVA degradation and without an RBS. B0015 is a double terminator, or a combination of two terminators: B0010 and B0012. We were inserting INSERT1 in all plasmids transformed into E. coli in our project. Because we also included pLac in all inserts, we could control the timing of gene expression after the pLac by adding IPTG to the medium and inhibiting lac I repressor.
We constructed “INSERT2” to induce phytase self-circularization. This part consists of 10 biological parts. The sequence begins with pLac, or a part of R0010 which originally has the last couple nucleotides of C0012. pLac is followed by B0034 which is also a kind of RBS. After B0034, there is a sequence coding for signal peptides which helps transportation of phytase to the intermembrane space of E. coli where the protein works. Phytase is sandwiched in between three different corresponding pairs of sequences. Two corresponding linker sequences are adjacent to phytase, and two RADA-16’s, and two specific corresponding sequences which code for “cysteine modules” are positioned toward the ends of the insert. A linker sequence codes for repeated amino acid sequence, GGGS. RADA-16 is a self-assembling peptide (SAP) sequence which iGEM HokkaidoU made into the major theme for last year’s project. Cysteine module is a short sequence composed of cysteine, glycine, and serine, two corresponding modules form disulfide bonds. INSERT2 also includes a His-tag, or six consecutive histidines, necessary for phytase purification. Lastly, there is a terminator B0015.
“INSERT3” is for checking SAP’s contribution to the disulfide bond formation respectively between linkers and cysteine modules. The part construct is quite like that of part 2. The only difference is that part 3 lacks SAP coding sequences. In other words, cysteine modules are adjacent to the linkers respectively on both ends of phytase.
We constructed “INSERT4” to conduct a research on the characteristics of phytase itself in terms of its stability under several different conditions. For example, we will test phytase’s vulnerability under heat, extreme pH and enzymatic influence. The insert consists of pLac, B0034, signal peptides, phytase, His-tag, and B0015.