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Part:BBa_K1467102

Designed by: Cara Deal   Group: iGEM14_NRP-UEA-Norwich   (2014-09-24)

GoldenGate compatible BS3 promoter + 5

BS3 is a plant specific promoter which is induced by the TALE AvrBS3. The promoter is a useful tool in testing a system in which protein expression is activated in response to plant cell infection (in this case represented by presence of AvrBS3).


This part is compatible with the GoldenGate MoClo Assembly Standard as it is free from internal BsaI and BpiI recognition sequences.


TAL (transcription activator-like) Effectors are proteins that are naturally excreted by the Xanthamonas type III secretion system and bind to specific recognition sequences on host DNA, activating a protective plant cell response following infection. These TALEs are now engineered for use in research into crop disease.


The advantage of using this inducible promoter alongside the TALE AvrBS3 is that it means real pathogens are not required to induce activation of the promoter BS3, allowing safe and easy testing of proof of concept systems in the laboratory. The presence of the AvrBS3 TALE effector represents infection of the plant by a pathogen- the BS3 promoter can be induced by this response, meaning that it is a useful tool in producing a response to pathogen infection.


iGEM14_NRP-UEA-Norwich have used this part to express reporter proteins when the construct is in the presence of the TALE AvrBS3. The aim of this was to test our system in which the presence of the pathogen (in this case the TALE AvrBS3) switches on the BS3 promoter, driving transcription of the coding sequence and therefore expression of the reporter protein of interest. These reporter proteins included GFP, Bax, NbHB1, AmilCP and AmilGFP. In constructs containing the promoter independent of AvrBS3, no transcription of the reporter protein occurred giving evidence that this part functions as expected. Examples of the promoter inducing GFP expression are shown below.


BS3 GFP + AvrBS3.jpg

Figure 1: A Nicotiana benthamiana leaf under UV light following infiltration (red circles) with Agrobacterium tumefaciens carrying plasmids encoding BS3_GFP_ocs (B) and BS3_GFP_ocs + 35s_AvrBS3_nos (A) respectively.

There is GFP expression in the area infiltrated with the multigene construct containing both the BS3 promoter and the TALE AvrBS3, showing that the promoter is active (A). In the area in which the infiltrated agrobacterium was just carrying the transcriptional unit encoding the promoter, GFP and terminator, there is no expression of GFP as the BS3 promoter is not active (B). This shows the function of AvrBS3 in activating the inducible BS3 promoter.


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]


[edit]
Categories
//chassis/eukaryote/nbenthamiana
//promoter
//regulation
Parameters
n/aGoldenGate compatible BS3 promoter + 5' UTR