Device

Part:BBa_K1913015

Designed by: Linea Katharina Muhsal   Group: iGEM16_Wageningen_UR   (2016-09-28)


Cry3Aa with araC/pBAD

Description

Cry3Aa with RBS, TEV-site, and His-tag. araC/pBAD in front of it. This BioBrick enables expression of the Cry toxin Cry3Aa1. Expression is inducible by addition of arabinose. After the Cry3Aa coding sequence, a TEV-site and a His-tag are added, enabling His-purification and then cutting off the His-tag to prevent interaction with the desired Cry3Aa protein.

T--Wageningen_UR--LMBB.jpg
Figure 1: Schematic overview of elements contained in this BioBrick.


Sequence and Features

Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal prefix found in sequence at 1219
    Illegal SpeI site found at 3266
    Illegal PstI site found at 3280
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1219
    Illegal NheI site found at 1205
    Illegal SpeI site found at 3266
    Illegal PstI site found at 3280
    Illegal NotI site found at 1225
    Illegal NotI site found at 3273
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1219
    Illegal BamHI site found at 1144
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found in sequence at 1219
    Illegal suffix found in sequence at 3266
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found in sequence at 1219
    Illegal XbaI site found at 1234
    Illegal SpeI site found at 3266
    Illegal PstI site found at 3280
    Illegal AgeI site found at 979
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal EcoRI site found at 1219
    Illegal XbaI site found at 1234
    Illegal SpeI site found at 3266
    Illegal PstI site found at 3280

To prove the Cry3Aa toxin can be expressed using E. coli BL21 with induction of 1 % arabinose, cells were grown and both soluble and insoluble proteins were run on an SDS-PAGE. A band with the corresponding size can be found in the pellet proteins.

T--Wageningen_UR--Cry3AafromEcoli.jpg
Figure 2: SDS-PAGE gel. M represents the marker lane. In lane 1 and 2 is the supernatant of the protein extract is visible and lane 3 and 4 represent the pellet. Lane 2 and 4 are the negative controls, whereas the protein extracts from lane 1 and 3 originate from transformant. The expected size of the Cry3Aa_TEV_HIs protein is 76 kDa.

Cry3Aa is known to be toxic to Tenebrio molitor. Therefore, we tested the toxicity of the toxin expressed with E. coli BL21 with an in vitro toxicity assay.

T--Wageningen_UR--boxplotjacco.jpg
Figure 3: This boxplot shows the toxicity towards T, molitor of samples containing the expressed Cry3Aa protein compared to a negative control.


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