Plasmid

Part:BBa_K5218023

Designed by: Amy Fu, Robert, Lichuan Chen, Xiaojuan Wang   Group: iGEM24_BGI-MammothEdu-South   (2024-09-03)


p1300-pGRF3-RtTAL-GFP

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

Lengths：13524 bp

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

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

Characterisation

The RtTAL CDS is described in basic part BBa_K5218005; 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 repressed in pS1300-GFP control lines compared to water treatment, whereas the pS1300-RtTAL-GFP and p1300-pGRF3-RtTAL-GFP lines could compensate the repression. The results showed that RtTAL could stably enhance the bioluminescence of plants, and GRF3 has some level of responsiveness to HCHO. The enhancement of light intensity by GRF3 promoter is limited though. It is worth memtioning that the batch of tobacco materials for this experiment went through significant injury stress in the process of injection, which may affected the bioluminescence analysis and interpretation. Unfortunately, due to limited time, we did not conduct a second test on new batch of plants.

Figure 3. pGRF3-RtTAL in response to HCHO.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 1203
    Illegal XbaI site found at 2804
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 222
    Illegal BglII site found at 1746
    Illegal BglII site found at 2060
    Illegal BglII site found at 2093
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 1203
    Illegal XbaI site found at 2804
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 1203
    Illegal XbaI site found at 2804
  • 1000
    COMPATIBLE WITH RFC[1000]
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