Part:BBa_K5218012

pS1300-NtC3H-GFP

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

Lengths：12263 bp

Usage：Functional characterization of NtC3H CDS in transgenic tobacco.

The NtC3H CDS is described in basic part BBa_K52180011. 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-NtC3H-GFP was transformed into competent E. coli TOP10 cells and verified through colony PCR and sequencing.

In the construct, NtC3H 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-NtC3H-GFP construct.

Characterisation

The cloning strategy for NtC3H-GFP fusion gene is as follows: two rounds of primers pairs were designed. The first round reverse primer is located on the 3' UTR of NtC3H CDS for specificity. The second round PCR uses first round product as template, and the second reverse primer is located before the NtC3H CDS stop codon, and recombination overhang is added for cloning.

The non-stop-codon NtC3H CDS was amplified through 2 rounds of PCR, purified and cloned into pS1300-GFP vector via Gibson Cloning system. In the construct, NtC3H CDS, driven by a super promoter (CaMV 35S), is fused with eGFP reporter gene (3' end) followed by NOS terminator. The recombinant plasmid pS1300-NtC3H-GFP was transformed into E. coli TOP10 competent cells and verified through colony PCR and sequencing.

Figure 2. Cloning of NtC3H CDS into pS1300-GFP expression vector.

A. NtC3H CDS first round PCR product on agarose gel electrophoresis. Lane M, 1kb plus DNA ladder; first round PCR product (with stop codon, no recombination overhang) lane1, NtC3H.

B. Single colonies of pS1300-NtC3H-GFP transformants on LB kanamycin+ plate.

C. Colony PCR product on agarose gel electrophoresis. lane 1-12, 12 single colonies tested.

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 NtC3H in FBP.

The pS1300-NtC3H-GFP construct was transformed into Agrobacterium GV3101 strain, followed by transient transformation of tobacco leaf through injection. The transgenic tobacco leaves were examined under the fluorescence microscope. The results showed that NtC3H-GFP fusion protein signal was observed in the tobacco leaf epidermal cells, suggesting the engineering success of the construct.

Figure 3. Candidate gene-GFP fusion proteins expression in tobacco leaf epidermal cells.

The pS1300-NtC3H-GFP transgenic tobacco plants were also photographed in the dark room to record their bioluminescence intensity. Since the team could not get access to the plant in vivo imaging system, we used greyscale value of photos instead and quantified the data using ImageJ software. The results suggested that the pS1300-NtC3H-GFP lines were more brighter compared to the control lines.

Figure 4. Enhanced bioluminescence of pS1300-NtC3H-GFP transgenic lines.

A. Picture of control and pS1300-NtC3H-GFP lines in the light. Control:FBP-22 only; vector control: FBP-22 transformed with pS1300-GFP.

B. Picture of control and pS1300-NtC3H-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 plotted. The result showed that NtC3H CDS trangenic lines have higher bioluminescence intensity compared to the control lines, indicating its positive role on FBP manipulation.

Figure 5. Bioluminescence intensity of control and transgenic lines.

Greyscale value was used to represent bioluminescence intensity.

Figure 6. 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 7. Key components of caffeic acid pathway tested in our project.

Figure adopted from L. Li et al, 2021[2]

Reference

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
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 761
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 749