Difference between revisions of "Part:BBa K2915225"
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<partinfo>BBa_K2915225 short</partinfo> | <partinfo>BBa_K2915225 short</partinfo> | ||
| − | The TAL1 is a TALEN which came from a plant pathogenic bacteria in the genus Xanthomonas. | + | 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. |
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<!-- Add more about the biology of this part here | <!-- Add more about the biology of this part here | ||
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| − | <span class='h3bb'>Sequence and Features</span> | + | <span class='h3bb'>'''Sequence and Features'''</span> |
<partinfo>BBa_K2915225 SequenceAndFeatures</partinfo> | <partinfo>BBa_K2915225 SequenceAndFeatures</partinfo> | ||
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| − | ''' | + | ===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 | ||
| + | |||
| + | [[File:T--Aix-Marseille--TAL2 production picture.jpg|center]] | ||
| + | |||
| + | |||
| + | '''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. | ||
| + | |||
| + | [[File:T--Aix-Marseille--TAL2 production 1 picture.png|center|900px]] | ||
| + | |||
| + | |||
| + | '''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. | ||
| + | |||
| + | [[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. For more details see [https://2019.igem.org/Team:Aix-Marseille/Experiments experiments] page. | ||
| + | |||
| + | |||
| + | ===Design Notes=== | ||
| − | This biobrick is in RFC | + | This biobrick is in [https://parts.igem.org/Help:Standards/Assembly/RFC10 RFC 10] standards with: |
The prefixe (with ATG in frame): 5' GAATTC GCGGCCGC T TCTAGA TG GCCGGC | The prefixe (with ATG in frame): 5' GAATTC GCGGCCGC T TCTAGA TG GCCGGC | ||
| Line 30: | Line 64: | ||
- with a His-tag and a TEV site | - with a His-tag and a TEV site | ||
| − | - with a promotor T7 and RBS (BBa_K525998) which is the regulator. | + | - with a promotor T7 and RBS ([[Part:BBa_K525998|BBa_K525998]]) which is the regulator. |
Latest revision as of 21:48, 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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.