Difference between revisions of "Part:BBa K2915225"

Revision as of 21:46, 21 October 2019

IPTG inducible promoter (T7) with RBS TAL1 6-His-Tag with TEV site

The TAL1 is a TALEN which came from a plant pathogenic bacteria in the genus Xanthomonas. These designed proteins are able to bind itself specifically onto a DNA fragment ( 5'CTGATC3') of a Tubercolusis gene . The promoter T7 and RBS (BBa_K525998) are regulator that are assembled with TAL1, which allows the production TAL1 with its HisTag using E.coli DH5-alpha/BL21 E3 bacteria. We also tested production of the protein and used the His-Tag to purify the protein.

Sequence and Features

Experiments

IPTG inducible promoter (T7) with RBS – TAL1-HisTag- TEV site

The promoter T7 and RBS (Bba_K525998) are regulator that are assembled with TAL1, which allows the production TAL1 with its HisTag using E.coli DH5-alpha bacteria. This part can bind itself with a DNA sequence of 5’ CTGATC 3’ (BM1). This engineered TAL consist of repeat domain has a primary structure with two amino acids that differs in each domain, those two amino acids are a code for one nucleotide of the binding sequence selected. HD, NG, NN, NI, NG and HD are the two amino that are changed in the CTGATC sequence, respectfully.

Usage and Biology

This Transcription Activator-Like (TAL) is a specific DNA binding protein, that is designed to recognize a specific DNA binding motif (BM1). This BM1 are present on the DNA that are amplify by PSR.

Production

To verify the production of TAL1, an SDS PAGE was performed and stained with Coomassie blue Different growing conditions were tested to determine the best growing conditions. The production was also tested two types E.coli bacteria : -DH5a -BL21 DE3

Figure 1. SDS-PAGE of the production in DH5a of TAL1, well 1, 2 and 3 are not induced of TAL1, TAL2 clone1 and TAL2 clone2, respectively. Well 4, 5 and 6 are induced at 30°C with 0.1M IPTG for 3 hours of TAL1, TAL2 clone1 and TAL2 clone2, respectively.

Fig 2. SDS-PAGE of the production in BL21 DE3 of TAL1. Well 1, 3 and 5 are induced at 30°C with 0.1M IPTG for 3 hours of TAL1, TAL2 clone1 and TAL2 clone2, respectively. Well 2,4 and 6 are induced at 16°C with 0.1M IPTG overnight.

Purification

We were able to purify our protein using the HisTag on the C-terminal of our protein. We used a histidine tagged protein purification columns. To verify our purification, we performed a Western Blot analysis with a Red Ponceau staining.

Fig 3. Western Blot analysis of TAL1 after purification with HisTag column. LD= loading sample, NR= non-restraint, E1 to E5 correspond to the eluted fractions. For more details see experiments page.

Design Notes

This biobrick is in RFC 10 standards with:

The prefixe (with ATG in frame): 5' GAATTC GCGGCCGC T TCTAGA TG GCCGGC  and the suffixe (contain Stop in frame): ACCGGT TAAT ACTAGT A GCGGCCG CTGCAG 3'

This TAL1 exists with:

- with a His-tag and a TEV site

- with a promotor T7 and RBS (BBa_K525998) which is the regulator.