Part:BBa_K4729707

Ptac + VirG TiBo542 N80D + repABCa

Introduction

Agrobacterium mediated transformation is one of the most prolific methods for plant engineering, by far the most common method for iGEM teams as well. However, working with plant Synbio is still far from a straightforward endeavor. This year, we set our sights on creating molecular tools that facilitate achieving successful transformation in non-model plant species, one of them is our Best Composite Part.

In nature, members of the genus Agrobacterium (Aphaproteobacteria) are soil-borne plant pathogens that integrate hormone producing genes in the host plant genome to cause growth deformities and tumors. This mechanism can be explored by substituting the oncogenes by genes of interest, thus integrating them in the plant chromosomes. The most well known strain, Agrobacterium tumefaciens, promotes crown-gall disease in most dicotyledonous plants, however, other strains such as Agrobacterium rhizogenes are also able to transform plant cells (Bahramnejad et al., 2019; Barton et al., 2018). Despite their wide use, Agrobacterium mediated plant transformation is still only well established for a handful of model organisms, which makes it hard for iGEM teams to engineer local species.

Part of the problem lies in the high specificity between plants and the necessary Agrobacterium strain for transformation. As we found out, getting hold of the correct Agrobacterium strain for a plant species (if one is even known) is extremely hard, reaching up to hundreds of Euros and months of shipping time. This is made even worse by the confusing and often conflicting nomenclature of existing strains (De Saeger et al., 2021).

Therefore, we created a composite part that aims to improve the efficiency and host range of Agrobacterium strains by leveraging the virG transcription factor as a Master-Switch plant transformation. With it, we hope to enable future teams to further extend the garden of plant projects in iGEM.

The problem

The virG Master-Switch construct

Cross-talk assay

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 3840
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 4468
  Illegal PstI site found at 3840
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1741
  Illegal BamHI site found at 262
  Illegal BamHI site found at 1917
  Illegal BamHI site found at 2209
  Illegal XhoI site found at 227
  Illegal XhoI site found at 3480
  Illegal XhoI site found at 3834
  Illegal XhoI site found at 7176
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 3840
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 3840
  Illegal NgoMIV site found at 111
  Illegal NgoMIV site found at 2054
  Illegal NgoMIV site found at 3894
  Illegal NgoMIV site found at 4119
  Illegal NgoMIV site found at 4224
  Illegal NgoMIV site found at 5254
  Illegal AgeI site found at 5733
  
• 1000
  COMPATIBLE WITH RFC[1000]