Composite

Part:BBa_K5223005

Designed by: Chong Yao   Group: iGEM24_SCU-China   (2024-10-01)


Crocin Synthesis System in Nicotiana benthamiana

Biology and Usage

In designing crocin synthesis, we conducted codon optimization for four essential genes (GjCCD4a_opt, GjALDH2C3_opt, GjUGT74F8_opt, and GjUGT94E13_opt) specifically for Nicotiana benthamiana and transiently transfected them to facilitate crocin production in this plant. In previous studies, those four genes were mainly transfected into Nicotiana benthamiana by stable transfection. Theoretically, the crocin content in the improved Nicotiana benthamiana obtained by transient transfection will be higher. We also successfully verified the feasibility of transient transfer of these four genes into Nicotiana benthamiana, which can synthesize crocin successfully.

Crocin Synthesis System in Nicotiana benthamiana
Function Crocin Synthesis
Use in Nicotiana benthamiana
Backbone pEAQ-HT-DEST1
Derived from Gardenia jasminoides

Table 1 Biology and usage

Design and Properties:


Fig.1: Schematic Diagram of Gene Circuit

Fig.2: Expression Plasmid of GjUGT94E13_opt, GjUGT74F8_opt and GjCCD4a_opt

Fig.3: GjALDH2C3_opt Expression Plasmid

Experimental Approach:

(1) Plasmid vector construction (Take CCD4a as an example)
1. The CCD4a fragment was obtained from the puncture bacteria by PCR, and the target fragment was recombined to the entry plasmid pDONR207 by BP reaction.
2. pDONR207-CCD4a was chemically transformed into E. coli TOP10 and the bacterial solution was coated into LB culture with gentamicin and cultured at 37°C overnight.
3. Pick a single colony on the plate for amplification perform PCR bacterial testing, and send the PCR products containing the target band in the bacterial test to be sequenced;
4. Extract the plasmid containing the correct sequence of bacteria and recombine the target fragment on the entry plasmid to the expression plasmid pEAQ-dest1 by LR reaction;
5. pEAQ-dest1-CCD4a was chemically transformed into E. coli TOP10 and the bacterial solution was coated into LB culture with kanamycin and cultured overnight at 37°C.
6. Pick a single colony on the plate for amplification perform PCR bacterial testing, and send the PCR products containing the target band in the bacterial test to be sequenced;
7. Extract the plasmid containing the correct sequence of bacteria, chemically transform the plasmid into Agrobacterium GV3101, and coat the bacterial solution onto LB plates containing kanamycin and rifampicin, and incubate at 28°C for 24-48h;
8. Pick a single colony for amplification and perform PCR bacterial testing, send the PCR product containing the target band in the bacterial test to sequencing, and transfer the Agrobacterium detected with the correct sequence into 20% glycerol and refrigerate at -80 degrees Celsius.

(2) Plasmid vector construction (GjALDH2C3_opt: Gibson assembly)
1. Amplify target fragments using PCR with primers that include the overlapping sequences.
2. Purify PCR products using a gel extraction.
3. ClonExpress Ultra One Step Cloning Kit: Add the recommended volume as per the manufacturer's instructions.
4. After the reaction, directly transform E. coli TOP10 with the assembly reaction mix.
5. The remaining steps are the same as above.

(3) Agrobacterium transfection (Take CCD4a as an example)
1. Take the cryopreserved bacteria scribble the culture, and carry out PCR bacterial test;
2. Pick the bacteria containing the target band and transfer them to 6ml of LB culture medium containing kanamycin and rifampicin and shake for 24 h;
3. Pipette 10ul of Agrobacterium containing CCD4a for 24 hours and shake it to an OD600 of 0.8-1 in 50ml LB medium containing kanamycin and rifampicin;
4. Prepare the resuspension: Each 100mL or so resuspension consists of 90mL of ddH2O, 10mL of MES stock solution, 1mL of MgCl2 stock solution and 100μL of AS stock solution.
5. Pre-cool the 50ml centrifuge and centrifuge at 4000r at 4°C for 10min.
6. Pour off the medium, add 25mL of ddH2O, and swirl and shake evenly.
7. Centrifuge at 4000r for 10min at 4°C and pour ddH2O.
8. Add 15ml of resuspension and incubate in the dark for 2h.
9. After 2 hours of incubation, Agrobacterium containing CCD4a was mixed with other Agrobacterium in equal proportions and injected into the leaves of Benthamian tobacco through a syringe.

(4) Crocin LC-MS/MS Result
We plotted a standard curve for crocin I using LC-MS/MS results.

Fig.4: Crocin I standard curve

Our samples were divided into four groups: Wild-type control, CR-nbhqt2 control, Wild-Type experimental, and CR-nbhqt2 experimental. The WT experimental group couldn't meet detection standards, so we mixed samples, resulting in one value, which serves as the theoretical average for a one-sample t-test.Analysis (Fig 5) showed that crocin I was undetectable in the WT and CR-nbhqt2 control groups but present in the experimental groups. The CR-nbhqt2 experimental group exhibited significantly higher crocin content than the WT group, confirming successful transfection and expression of the crocin metabolome in Nicotiana benthamiana.

Fig.5: Crocin I LC-MS/MS result

Reference:

[1] Li S, Zhou Z, Li Y, Hu Y, Huang Z, Hu G, Wang Y, Wang X, Lou Q, Gao L, Shen C, Gao R, Xu Z, Song J, Pu X. Construction of a high-efficiency GjCCD4a mutant and its application for de novo biosynthesis of five crocins in Escherichia coli. Int J Biol Macromol. 2024 Oct;277(Pt 2):133985. doi: 10.1016/j.ijbiomac.2024.133985. Epub 2024 Jul 19. PMID: 39033887.
[2] Xie L, Luo Z, Jia X, Mo C, Huang X, Suo Y, Cui S, Zang Y, Liao J, Ma X. Synthesis of Crocin I and Crocin II by Multigene Stacking in Nicotiana benthamiana. Int J Mol Sci. 2023 Sep 15;24(18):14139. doi: 10.3390/ijms241814139. PMID: 37762441; PMCID: PMC10532124.
[3] Ji A, Jia J, Xu Z, Li Y, Bi W, Ren F, He C, Liu J, Hu K, Song J. Transcriptome-Guided Mining of Genes Involved in Crocin Biosynthesis. Front Plant Sci. 2017 Apr 11;8:518. doi: 10.3389/fpls.2017.00518. PMID: 28443112; PMCID: PMC5387100.
[4] Frusciante S, Diretto G, Bruno M, Ferrante P, Pietrella M, Prado-Cabrero A, Rubio-Moraga A, Beyer P, Gomez-Gomez L, Al-Babili S, Giuliano G. Novel carotenoid cleavage dioxygenase catalyzes the first dedicated step in saffron crocin biosynthesis. Proc Natl Acad Sci U S A. 2014 Aug 19;111(33):12246-51. doi: 10.1073/pnas.1404629111. Epub 2014 Aug 5. PMID: 25097262; PMCID: PMC4143034.
[5] Pu X, He C, Yang Y, Wang W, Hu K, Xu Z, Song J. In Vivo Production of Five Crocins in the Engineered Escherichia coli. ACS Synth Biol. 2020 May 15;9(5):1160-1168. doi: 10.1021/acssynbio.0c00039. Epub 2020 Apr 7. PMID: 32216376.

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 1678
    Illegal XhoI site found at 3405
    Illegal XhoI site found at 5753
    Illegal XhoI site found at 7618
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1402
    Illegal BsaI site found at 3129
    Illegal BsaI site found at 5477
    Illegal BsaI site found at 7342
    Illegal BsaI.rc site found at 1681
    Illegal BsaI.rc site found at 3408
    Illegal BsaI.rc site found at 5756
    Illegal BsaI.rc site found at 7621


[edit]
Categories
//chassis/eukaryote/nbenthamiana
Parameters
None