Difference between revisions of "Part:BBa K2915150"
 
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<partinfo>BBa_K2915150 parameters</partinfo>
 
<partinfo>BBa_K2915150 parameters</partinfo>
 
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=== Experiments===
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AFP-lu1 production We were able to insert the TAL2 gene into a plasmid containing the T7 promoter and RBS which is a psB1C3 vector. SDS-PAGE was performed and stained with Coomassie blue using cells containing this Biobrick, to verify the production of our protein we did different growing conditions to determine the best growing conditions by using two types E.coli bacteria : -DH5a -BL21 DE3
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[[File:T--Aix-Marseille--AF-lu production picture.png|link=]]
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Figure 1. SDS-PAGE of the production in DH5a and BL21 DE3 of AF-lu1, well 1 and 2 are induced at 30°C with 0.1M IPTG for 3hours in DH5a and BL21 DE3, respectively. Well 5 and 6 are not induced of AF-lu1 in DH5a and BL21 DE3 respectively. Well 3 and 4 are induced at 16°C with 0.1M IPTG overnight in DH5a and BL21 DE3, respectively.
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AFP-lu1purification After having realized the AFP-lu1 production tests, the purification was realized in order to obtain pure protein fractions. For this, the E. coli BL21 cells were lysates and then are purified. Purification of the protein was realized with a HisTag column which is a manual chromatography system for quick, easy ad small quantity purification. The fusion of the histine tag with AFP-lu1 allows to separate our protein from the other proteins.
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[[File:T--Aix-Marseille--AF-lu purification picture.png|link=]]
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Fig 2. SDS-PAGE of AFP-lu1 after purification with HisTag column. LD= loading sample, NR= non-restraint, E1 to E7 correspond to the eluted fractions.
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[[File:T--Aix-Marseille--AF-lu Western blot picture.png|link=]]
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Fig 3. Western Blot analysis of AFP-lu1 after purification with HisTag column. (a) LD= loading sample, NR= non-restraint, E1 to E3 correspond to the eluted fractions. (b) from E4 to E7 correspond to the eluted fractions.
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See more...
  
 
===Design Notes===
 
===Design Notes===

Latest revision as of 18:04, 21 October 2019


IPTG inducible promoter (T7) with RBS AFP-lu1 6-His-Tag with TEV site

The AFP-Iu1 it's a antifreeze which came from the Choristoneura fumiferana. These proteins lower the nonequilibrium freezing point of water while not affecting the melting point. The AFP proteins were used in our project in order to conserved all engeneering proteins we designed to create our TB diagnostic Kit.The AFP protein was produced and purified because of the presence of a 6His-Tag in the N-terminal of the protein.

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
    COMPATIBLE WITH RFC[1000]


Experiments

AFP-lu1 production We were able to insert the TAL2 gene into a plasmid containing the T7 promoter and RBS which is a psB1C3 vector. SDS-PAGE was performed and stained with Coomassie blue using cells containing this Biobrick, to verify the production of our protein we did different growing conditions to determine the best growing conditions by using two types E.coli bacteria : -DH5a -BL21 DE3

T--Aix-Marseille--AF-lu production picture.png

Figure 1. SDS-PAGE of the production in DH5a and BL21 DE3 of AF-lu1, well 1 and 2 are induced at 30°C with 0.1M IPTG for 3hours in DH5a and BL21 DE3, respectively. Well 5 and 6 are not induced of AF-lu1 in DH5a and BL21 DE3 respectively. Well 3 and 4 are induced at 16°C with 0.1M IPTG overnight in DH5a and BL21 DE3, respectively.


AFP-lu1purification After having realized the AFP-lu1 production tests, the purification was realized in order to obtain pure protein fractions. For this, the E. coli BL21 cells were lysates and then are purified. Purification of the protein was realized with a HisTag column which is a manual chromatography system for quick, easy ad small quantity purification. The fusion of the histine tag with AFP-lu1 allows to separate our protein from the other proteins.

T--Aix-Marseille--AF-lu purification picture.png

Fig 2. SDS-PAGE of AFP-lu1 after purification with HisTag column. LD= loading sample, NR= non-restraint, E1 to E7 correspond to the eluted fractions.

T--Aix-Marseille--AF-lu Western blot picture.png


Fig 3. Western Blot analysis of AFP-lu1 after purification with HisTag column. (a) LD= loading sample, NR= non-restraint, E1 to E3 correspond to the eluted fractions. (b) from E4 to E7 correspond to the eluted fractions.

See more...

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.