Difference between revisions of "Part:BBa K1789006"
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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. | 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 | + | 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’ | BM1: 5’-GGAGGCACCGGTGG-3’ | ||
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[[File:SCAF2_A.jpg|500px|]] | [[File:SCAF2_A.jpg|500px|]] | ||
| − | This scaffold is designed to put enzymes | + | This scaffold is designed to put enzymes in the same direction. A 6-bp intervening sequence is added between two BMs. |
==Sequence and Features== | ==Sequence and Features== | ||
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===ChIP-PCR Analysis=== | ===ChIP-PCR Analysis=== | ||
| − | To evaluate whether SCAF2 can effectively target the binding motifs on plasmid DNA scaffold, the ChIP-PCR analysis was conducted. For this experiment, the plasmid of pSB1C3-Plac-RBS-TALE2-GFP2-Ter-Scaffold2 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 P3 and reverse P4 for GFP2 amplification (Fig. 1). | + | To evaluate whether SCAF2 can effectively target the binding motifs on plasmid DNA scaffold, the ChIP-PCR analysis was conducted. For this experiment, the plasmid of pSB1C3-Plac-RBS-TALE2-GFP2-Ter-Scaffold2 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 P3 and reverse P4 for GFP2 amplification (Fig. 1). |
[[File:TALE2 A.jpg|500px|]] | [[File:TALE2 A.jpg|500px|]] | ||
Latest revision as of 02:20, 19 September 2015
SCAF2
This part is a highly repetitive DNA sequence which can bind TALE protein specifically.
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 in the same direction. A 6-bp intervening sequence is added 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 50
Illegal AgeI site found at 92
Illegal AgeI site found at 134
Illegal AgeI site found at 176
Illegal AgeI site found at 218
Illegal AgeI site found at 260 - 1000COMPATIBLE WITH RFC[1000]
Experimental Validation
Sequencing
This part is sequenced as correct after construction.
ChIP-PCR Analysis
To evaluate whether SCAF2 can effectively target the binding motifs on plasmid DNA scaffold, the ChIP-PCR analysis was conducted. For this experiment, the plasmid of pSB1C3-Plac-RBS-TALE2-GFP2-Ter-Scaffold2 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 P3 and reverse P4 for GFP2 amplification (Fig. 1).
Fig. 1 A schematic showing the primers and the plasmid regions tested in ChIP assays. P3/P4 was designed for TALE2-GFP2 ChIP assay.
As shown in Fig 2, a 251 bp of DNA fragments was amplified from the precipitates of TALE2-GFP2-Scaffold2 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 SCAF2 can be specifically targeted and combined by corresponding TAL effectors in vivo.
Fig. 2 Determination of the binding abilities of TALE2-GFP2 to corresponding DNA scaffolds. Input indicates an aliquot of total DNA. Antibodies used for immunoprecipitation are indicated above the lanes.