Plasmid

Part:BBa_K5218004

Designed by: Rui Zhang, Lichuan Chen, Xiaojuan Wang   Group: iGEM24_BGI-MammothEdu-South   (2024-08-18)


pS1300-RcTAL-GFP

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

Lengths：12326 bp

Usage：Functional characterization of RcTAL CDS in transgenic tobacco.


The RcTAL CDS is described in basic part BBa_K5218001. The CDS sequence was cloned into pS1300-GFP vector via Gibson Cloning system. pS1300-GFP is a pCAMBIA1300 backbone plasmid with 8613 bp length. The recombinant plasmid pS1300-AtC3H-GFP was transformed into competent E. coli TOP10 cells and verified through colony PCR and sequencing.

In the construct, RcTAL CDS,driven by a super promoter (CaMV 35S), is fused with eGFP reporter gene (3' end) followed by NOS terminator. The plasmid was transformed into Agrobacterium GV3101 strain for gene functional validation.

Figure 1. Map of pS1300-RcTAL-GFP construct.


Characterisation

Nicotiana benthamiana tobacco line FBP-22[1], in which the Fungal Bioluminescence Pathway (FBP, includes LUZ, H3H, CPH and HispS gene) was introduced, was proven to be autoluminescent. Yet the luminescence intensity was limited. Team BGI-MammothEdu-South 2024 foucsed on enhancing bioluminescence of plants and exploring various applications. Tobacco line FBP-22 was used as control and genetical engineering material to verify the function of RcTAL in FBP.

pS1300-RcTAL-GFP construct was transformed into Agrobacterium GV3101 strain. Transient transformation of tobacco leaf epidermal cells was conducted. The transgenic tobacco leaves were examined under the fluorescence microscope. The result shows that RcTAL-GFP fusion protein was observed in the tobacco leaf epidermal cells, suggesting the engineering success of the construct.

Figure 2. Candidate gene-GFP fusion proteins expression in tobacco leaf epidermal cells.

The pS1300-RcTAL-GFP transgenic tobacco plants were also photographed in the dark room to record their bioluminescence intensity. It was clear from the photo that the pS1300-RcTAL-GFP lines were brighter than the control lines. Since the team could not get access to the plant in vivo imaging system, we used greyscale value of photos instead and quantified using ImageJ software.

Figure 3. Enhanced bioluminescence of pS1300-RcTAL-GFP transgenic lines.

A. Picture of control and pS1300-RcTAL-GFP lines in the light. Control:FBP-22 only; vector control: FBP-22 transformed with pS1300-GFP.
B. Picture of control and pS1300-RcTAL-GFP lines in the dark.
C. Grayscale values of randomly circled areas in A and B. Value calculated with ImageJ software.

All the grey-scale value data were collected from the lines tested and bar charts were plotted. The result showed that RcTAL CDS trangenic line had one of the highest bioluminescence intensity enhancement compared to the control lines, indicating its positive role on FBP manipulation.

Figure 4. Bioluminescence intensity of control and transgenic lines.
Greyscale value was used to represent bioluminescence intensity.

Figure 5. Bioluminescence intensity enhancement of different transgenic lines.
Increased greyscale value compared to vector control group.

Team BGI-MammothEdu-South 2024 focused on two genes, TALs and C3Hs from various species, tested their functions in the caffeic acid pathway. The results showed that the TAL and C3H genes we selected could all contribute to a range of bioluminescence intensity enhancement, presumably due to the FBP substance caffeic acid accumulation. These findings could provide valuable insights for future iGEM team or researchers in the field of bioluminescence plants.

Figure 6. Key components of caffeic acid pathway tested in our project.
Figure adopted from L. Li et al, 2021[2]

Reference

<p>1.Zheng, P., Ge, J., Ji, J., Zhong, J., Chen, H., Luo, D., Li, W., Bi, B., Ma, Y., Tong, W., Han, L., Ma, S., Zhang, Y., Wu, J., Zhao, Y., Pan, R., Fan, P., Lu, M. and Du, H. (2023), Metabolic engineering and mechanical investigation of enhanced plant autoluminescence. Plant Biotechnol J, 21: 1671-1681.

2.Li LL, Liu X, Qiu ZT, et al. (2021) Microbial synthesis of plant polyphenols. Chinese Journal of Biotechnology, 37(6): 2050-2076.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 490
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 490
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 490
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 490
    Illegal AgeI site found at 646
  • 1000
    COMPATIBLE WITH RFC[1000]


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