Difference between revisions of "Part:BBa K2915275"
Line 18: Line 18:
 
<partinfo>BBa_K2915275 parameters</partinfo>
 
<partinfo>BBa_K2915275 parameters</partinfo>
 
<!-- -->
 
<!-- -->
 +
 +
 +
 +
=== Experiments===
 +
 +
====IPTG inducible promoter (T7) with RBS – TAL2-HisTag- TEV site====
 +
 +
The promoter T7 and RBS (Bba_K525998) are regulator that are assembled with TAL2, which allows the production TAL2 with its HisTag using E.coli DH5-alpha bacteria.
 +
This part can bind itself with a DNA sequence of 5’ CCGCTG 3’ (BM2). 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, HD, NN, HD, NG and NN are the two amino that are changed in the CCGCTG 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 TAL2, 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
 +
 +
 
 +
[[File:T--Aix-Marseille--TAL2 production picture.jpg|center]]
 +
 +
'''Figure 1. SDS-PAGE of the production in DH5a of TAL''', 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.
 +
 +
[[File:T--Aix-Marseille--TAL2 production 1 picture.png|center|900px]]
 +
 +
'''Fig 2. SDS-PAGE of the production in BL21 DE3 of TAL'''. 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.
 +
 +
 +
[[File:T--Aix-Marseille--TAL2 WB purif picture.png|center|900px]]
 +
 +
'''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.
 +
 +
====Activity test====
 +
To verify the functionality of this part we did an electrophoretic mobility shift assay (EMSA) also none as a gel shift assay, which is a method that’s able to analyze the potential interaction protein-DNA. We used the gel shift assay in order to study our proteins for the respectively targeted DNA amplicons.
 +
 +
[[File:T--Aix-Marseille--TAL2 gel shift 2.png picture.png|center|300px]]
 +
 +
'''Fig 4. Activity test of TAL2 in the presence/absence of the DNA amplify.'''
 +
Description of the test activity for TAL2, see experiments page.
 +
 +
  
 
===Design Notes===
 
===Design Notes===

Revision as of 17:23, 21 October 2019


IPTG inducible promoter (T7) with RBS TAL2 6-His-Tag with TEV site

The TAL2 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'CCGCTG3') of a Tubercolusis gene . The promoter T7 and RBS (BBa_K525998) are regulator that are assembled with TAL2, which allows the production TAL2 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 and we tested the activity of our protein using a gel shift.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 246



Experiments

IPTG inducible promoter (T7) with RBS – TAL2-HisTag- TEV site

The promoter T7 and RBS (Bba_K525998) are regulator that are assembled with TAL2, which allows the production TAL2 with its HisTag using E.coli DH5-alpha bacteria. This part can bind itself with a DNA sequence of 5’ CCGCTG 3’ (BM2). 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, HD, NN, HD, NG and NN are the two amino that are changed in the CCGCTG 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 TAL2, 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


T--Aix-Marseille--TAL2 production picture.jpg

Figure 1. SDS-PAGE of the production in DH5a of TAL, 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.

T--Aix-Marseille--TAL2 production 1 picture.png

Fig 2. SDS-PAGE of the production in BL21 DE3 of TAL. 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.


T--Aix-Marseille--TAL2 WB purif picture.png

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.

Activity test

To verify the functionality of this part we did an electrophoretic mobility shift assay (EMSA) also none as a gel shift assay, which is a method that’s able to analyze the potential interaction protein-DNA. We used the gel shift assay in order to study our proteins for the respectively targeted DNA amplicons.

T--Aix-Marseille--TAL2 gel shift 2.png picture.png

Fig 4. Activity test of TAL2 in the presence/absence of the DNA amplify. Description of the test activity for TAL2, 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 TAL2 exists with:

- with a His-tag and a TEV site

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