Split T7 RNA polymerase Cterm conjugated to Trastuzumab with a soluble linker
Part for expression of the split T7 RNA polymerase Cterm conjugated to Trastuzumab with a soluble linker in PURE System
Sequence and Features
Assembly Compatibility:
10
INCOMPATIBLE WITH RFC[10]
Illegal XbaI site found at 40
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
INCOMPATIBLE WITH RFC[23]
Illegal XbaI site found at 40
25
INCOMPATIBLE WITH RFC[25]
Illegal XbaI site found at 40 Illegal NgoMIV site found at 1080 Illegal AgeI site found at 549
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 21
Introduction
Figure 1: Trastuzumab-SL-T7Cterm structure
The CALIPSO part BBa_K4768006 is composed of the anti-HER2 antibody Trastuzumab fused to the C-terminal subunit of the T7 RNA polymerase (residues 181 to 704) through a soluble linker. This gene is under transcriptional control of an SP6 promoter and T7 terminator.
This part, coupled to the part BBa_K4768005 containing the N-terminal subunit of the T7 RNA polymerase, has been designed to develop a split T7 RNAP-based biosensor capable of recognizing HER-2, an epidermal growth factor that is overexpressed in cancer cells [1], in solution.
The HER2-induced T7 RNAP complex was designed from two existing constructs: a split T7 RNAP-based biosensor for the detection of rapamycin [2] and a split luciferase conjugated with antibodies capable of recognizing HER2[3]. We decided to merge the relevant functionalities of these two constructs and created a new biosensor that transduces HER2 binding to gene expression activation.
Figure 2: Recognition of HER2 extracellular domain induces functional assembly of the split T7 RNA polymerase, which enables gene expression of target gene under control of a T7 promoter.
Construction
The CALIPSO part BBa_K4768006 consists in the C-terminal subunit of the T7 RNA polymerase fused to trastuzumab, an anti-HER2 antibody, on its C-terminal domain through an 8-amino-acid linker of glycine and serine residues. The synthesis of this gBlock was made by IDT. Finally, the gBlock was cloned into the pET21a (+) plasmid with Takara In-Fusion kit (In-Fusion® Snap Assembly Master Mix, 638948) and introduced into Stellar competent cells.
We cloned the gBlock in pET21 by using the following primers (from 5' to 3'):
Primer T7-F: agttcctcctttcagatttaggtgacactataggggagac
Primer T7-R: gagatctcgatcccgcaaaaaacccctcaagacccg
Figure 3Agarose gel electrophoresis or PCR screening on plasmids extracted from 24 clones.
24 transformants were screened by colony PCR with primers flanking the insertion site within pET21 ( using primers T7-F and T7-R). Unfortunately, no positive transformant was detected (fig. 3).
