Part:BBa_K1789005
SCAF1
This part is a scaffold system contains the recognizing motifs of TALE1 and TALE3
Usage and Biology
As is mentioned in our project description, different TALEs share a similar domain structure that enables them to bind the genome of the host cell and act as transcriptional effectors. By engineering those structures, we can build proteins that can bind with any DNA sequence that we desire.
In our project, we designed two different DNA binding motifs (DNA BMs). The sequences were chosen from Danio rerio CD154 gene in order to avoid homology with E.coli genome. Those BMs are sequenced as
BM1: 5’-GGAGGCACCGGTGG-3’
MB2: 5’-GATAAACACCTTTC-3’
Those sequences were repeated for more than 10 times in a plasmid, with different length of intervening sequence.
This scaffold is designed to put enzymes as close as possible, no intervening sequence is inserted between two BMs.
Sequence and Features
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 8
Illegal AgeI site found at 38
Illegal AgeI site found at 68
Illegal AgeI site found at 98
Illegal AgeI site found at 128
Illegal AgeI site found at 158
Illegal AgeI site found at 188 - 1000COMPATIBLE WITH RFC[1000]
Experimental Validation
Sequencing
This part is sequenced as correct after construction.
ChIP-PCR Analysis
To evaluate whether SCAF1 can be effectively targeted and combined by TALEs, the ChIP-PCR analysis was conducted. For this experiment, the plasmid of pSB1C3-RBS-TALE1-GFP1-Ter-Scaffold1 was constructed, interpret into E.coli BL21 (DE3), and subsequently induced expression by IPTG. Bacterial lysis samples were cross-linked in 1% formaldehyde without ultrasonic treatment due to the small size of binding plasmid, and immunoprecipitated with anti-GFP polyclonal antibody. Because the binding motifs of TALEs are containing highly repeated sequences, and their flanking sequences are also homologous to the other parts of the harboring plasmid, the primers used for ChIP-PCR were forward P1 and reverse P2 for GFP1 amplification (Fig. 1).
Fig. 1 A schematic showing the primers and the plasmid regions tested in ChIP assays. P1/P2 was designed for TALE1-GFP1 ChIP assay.
As shown in Fig 2, a 471 bp of DNA fragments was amplified from the precipitates of TALE1-GFP1-Scaffold1 using anti-GFP antibody. However, the negative control immunoprecipitations using no antibody (beads only) or normal rabbit IgG showed no amplification signal. The amplified fragment was confirmed by sequencing. These results indicate that SCAF1 can be specifically targeted and combined by corresponding TALE fused proteins in vivo.
Fig. 2 Determination of the binding abilities of TALE1-GFP1 to corresponding DNA scaffolds. Input indicates an aliquot of total DNA. Antibodies used for immunoprecipitation are indicated above the lanes.
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