Difference between revisions of "Part:BBa K759001"
 
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The wildtype Ag43 have 6 PstI sites.
 
The wildtype Ag43 have 6 PstI sites.
 
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==Optimum aggregation method==
 
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[[image: HokkaidoU2012 Ag43 Image8 .JPG|left|thumb|Image1 In plastic test tube, E. coli forms small cluster.]]
 
[[image: HokkaidoU2012 Ag43 Image8 .JPG|left|thumb|Image1 In plastic test tube, E. coli forms small cluster.]]
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Reciprocal shaking of culture flask seemed to be better than rotation.
 
Reciprocal shaking of culture flask seemed to be better than rotation.
 
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==Cultivating in glass tube is suitable for aggregation==
 
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Induction of function of this part was tested by observing appearance of aggregated E. coli cluster in the presence (right) or absence (left) of L-arabinose in culture medium (See Image2).  Aggregated E. coli clusters were observed only in the culture medium containing L-arabinose. The results suggest that the auto-aggregation of E. coli is mediated by overexpressed Ag43.
 
Induction of function of this part was tested by observing appearance of aggregated E. coli cluster in the presence (right) or absence (left) of L-arabinose in culture medium (See Image2).  Aggregated E. coli clusters were observed only in the culture medium containing L-arabinose. The results suggest that the auto-aggregation of E. coli is mediated by overexpressed Ag43.
 
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[[image: HokkaidoU2012 Ag43 Image12.jpg|center|thumb|Image2  Induction of function of [https://parts.igem.org/Part:BBa_K759012  BBa_K759012] (almost same part with this part without existence of eCFP coding site). Appearance of aggregated E. coli in the presence (right) or absence (left) of L-arabinose in culture medium. Plasmid structure is pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28 (low copy plasmid), and culture condition is at 37C for 24 hrs at 130 rpm.]]
 
[[image: HokkaidoU2012 Ag43 Image12.jpg|center|thumb|Image2  Induction of function of [https://parts.igem.org/Part:BBa_K759012  BBa_K759012] (almost same part with this part without existence of eCFP coding site). Appearance of aggregated E. coli in the presence (right) or absence (left) of L-arabinose in culture medium. Plasmid structure is pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28 (low copy plasmid), and culture condition is at 37C for 24 hrs at 130 rpm.]]
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[[image:HokkaidoU2012 Ag43 Image13.JPG|400px|center|thumb|Image3  Graph of OD600 of suspension and culture time (hours). ]]
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 15:51, 3 October 2012

Aggregation Module inducible by arabinose in E.coli.

In the presence of L-arabinose, pBAD promoter is induced and then wildtype Antigen 43 (Ag43) protein is produced.

The wildtype Ag43 have 6 PstI sites.

Optimum aggregation method

Image1 In plastic test tube, E. coli forms small cluster.

To aggregate cells each other, L-arabinose was added into 2ml LB liquid medium with appropriate antibiotics for induction of the function of this part, and cultured in plastic test tubes. The plastic tube was not suitable for producing large cluster of E. coli cells, since a strong affinity of E. coli expressing Ag 43 was observed onto the surface of inside of plastic test tube.

Meanwhile, we tested the same observation using 300 ml Erlenmeyer flask containing 50 ml LB liquid medium and cultivated at 34C (See [http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/aggregation_protocols iGEM12_HokkaidoU_Japan wiki protocol page] for experimental procedure)

We found that optimum temperature required for efficient aggregation by this part is 34~37C. We didn’t observe aggregation at 30C even after 24 hr-culture. Reciprocal shaking of culture flask seemed to be better than rotation.



Cultivating in glass tube is suitable for aggregation

Induction of function of this part was tested by observing appearance of aggregated E. coli cluster in the presence (right) or absence (left) of L-arabinose in culture medium (See Image2). Aggregated E. coli clusters were observed only in the culture medium containing L-arabinose. The results suggest that the auto-aggregation of E. coli is mediated by overexpressed Ag43.

Image2 Induction of function of BBa_K759012 (almost same part with this part without existence of eCFP coding site). Appearance of aggregated E. coli in the presence (right) or absence (left) of L-arabinose in culture medium. Plasmid structure is pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28 (low copy plasmid), and culture condition is at 37C for 24 hrs at 130 rpm.
Image3 Graph of OD600 of suspension and culture time (hours).


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1205
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1144
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1945
    Illegal NgoMIV site found at 3169
    Illegal NgoMIV site found at 3679
    Illegal NgoMIV site found at 3700
    Illegal AgeI site found at 979
    Illegal AgeI site found at 2695
    Illegal AgeI site found at 3439
    Illegal AgeI site found at 3853
    Illegal AgeI site found at 4095
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 961