Difference between revisions of "Part:BBa K2915225:Experience"
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| − | + | ===Applications of BBa_K2915225 : TAL1=== | |
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| − | ===Applications of BBa_K2915225=== | + | |
====IPTG inducible promoter (T7) with RBS – TAL1-HisTag- TEV site==== | ====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. | 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. | ||
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-BL21 DE3 | -BL21 DE3 | ||
| − | [[]] | + | [[File:T--Aix-Marseille--TAL2 production picture.jpg|center]] |
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'''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. | '''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]] |
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'''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. | '''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. | ||
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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. | 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]] |
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| − | '''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. | + | '''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. |
===User Reviews=== | ===User Reviews=== | ||
Latest revision as of 12:18, 18 October 2019
Applications of BBa_K2915225 : TAL1
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.
User Reviews
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