Part:BBa_K5218024

p1300-pGRF3-AtC3H-GFP

Source：plasmid derived from pCAMBIA1300 backbone with Gibson Cloning system.

Lengths：12919 bp

Usage：Functional characterization of pGRF3-AtC3H module in transgenic tobacco.

Figure 1. p1300-pGRF3-AtC3H-GFP plasmid map.

Characterisation

The AtC3H CDS is described in basic part BBa_K5218002; GRF3 promoter is described in basic part BBa_K5218017. This combination integrates a formaldehyde-responsive regulatory module with a luminescence-enhancing gene to create luminescent plants that respond to formaldehyde.

Figure 2. Generation of p1300-pGRF3-RtTAL-GFP and p1300-pGRF3-AtC3H-GFP construct.

A. PCR product on agarose gel electrophoresis. Lane M, 1kb plus DNA marker; lane 1&2, MDH1 promoter; lane 3&4, GS1 promoter; lane 5&6, GRF3 promoter. Lane 1/3/5 products contain RtTAL recombination overhang; lane 2/4/6 products contain AtC3H recombination overhang;

B. Single colonies of p1300-pGRF3-RtTAL-GFP transformants on LB kanamycin+ plate.

C. Single colonies of p1300-pGRF3-AtC3H-GFP transformants on LB kanamycin+ plate.

D. Single colonies of p1300-pGRF3-RtTAL-GFP transformants on LB kanamycin+ rifampicin+ plate.

E. Single colonies of p1300-pGRF3-AtC3H-GFP transformants on LB kanamycin+ rifampicin+ plate.

F. Colony PCR of p1300-pGRF3-RtTAL-GFP in E.coli. Lane M, 5000bp DNA marker. Lane 1-24, 24 single colonies tested.

G. MDH1 promoter sequence validated through sequencing.

H. Colony PCR of p1300-pGRF3-AtC3H-GFP in E.coli. Lane M, 5000bp DNA marker. Lane 1-24, 24 single colonies tested.

I. MDH1 promoter sequence validated through sequencing.

J. Colony PCR of p1300-pGRF3-RtTAL-GFP in GV3101. Lane M, 5000bp DNA marker. Lane 1-8, 8 single colonies tested.

K. Colony PCR of p1300-pGRF3-AtC3H-GFP in GV3101. Lane M, 5000bp DNA marker. Lane 1-8, 8 single colonies tested.

The p1300-pGRF3-RtTAL-GFP and p1300-pGRF3-AtC3H-GFP constructs were transformed into Agrobacterium GV3101 strain, followed by infiltration to generate transient expression lines.

Upon 2mM HCHO treatment for 36 hours, the bioluminescence intensity was clearly repressed in pS1300-GFP control lines, whereas the pS1300-AtC3H-GFP and p1300-pGRF3-AtC3H-GFP lines could compensate the repression. The results showed that AtC3H could stably enhance the bioluminescence of plants, and GRF3 is indeed responsive to HCHO. Yet the enhancement of light intensity by promoter GRF3 is limited.

Figure 3. pGRF3-AtC3H in response to HCHO.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 1203
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 2023
  Illegal NheI site found at 2341
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 222
  Illegal BglII site found at 1746
  Illegal XhoI site found at 2195
  Illegal XhoI site found at 2538
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 1203
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 1203
  Illegal AgeI site found at 1921
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 2982
  Illegal SapI site found at 2176