Composite

Part:BBa_K4399013

Designed by: Duoqing LIN   Group: iGEM22_Worldshaper-Shanghai   (2022-09-27)
Revision as of 04:47, 8 October 2022 by Htclamm (Talk | contribs)


The negative control for anthocyanin biosynthesis

It is used as a negative control for inducible anthocyanin biosynthesis study, which contains a constitutive GFP expression box, an inducible SbMYB75 expression box and an inducible SbDEL expression box. However, as there is not a XVE box, the activator for PLexA35S, so neither SbMYB75 nor SbDEL can be expressed.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1342
    Illegal EcoRI site found at 5513
    Illegal XbaI site found at 696
    Illegal XbaI site found at 1579
    Illegal PstI site found at 2759
    Illegal PstI site found at 4179
    Illegal PstI site found at 4264
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1342
    Illegal EcoRI site found at 5513
    Illegal NheI site found at 1244
    Illegal PstI site found at 2759
    Illegal PstI site found at 4179
    Illegal PstI site found at 4264
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1342
    Illegal EcoRI site found at 5513
    Illegal BglII site found at 4113
    Illegal BglII site found at 4925
    Illegal BamHI site found at 3547
    Illegal BamHI site found at 3849
    Illegal BamHI site found at 5168
    Illegal BamHI site found at 5237
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1342
    Illegal EcoRI site found at 5513
    Illegal XbaI site found at 696
    Illegal XbaI site found at 1579
    Illegal PstI site found at 2759
    Illegal PstI site found at 4179
    Illegal PstI site found at 4264
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1342
    Illegal EcoRI site found at 5513
    Illegal XbaI site found at 696
    Illegal XbaI site found at 1579
    Illegal PstI site found at 2759
    Illegal PstI site found at 4179
    Illegal PstI site found at 4264
  • 1000
    COMPATIBLE WITH RFC[1000]

Results

Construction of level-0 vectors

The DNA elements (golden gate compatible, promoters: PAtUBI5, PLexA35S, P2×35S; CDS of Genes: GFP, SbMYB75, SbDEL, XVE; Terminators: Tmas, Thsp18.2, Tnos, T35S) were synthesized by Gensript based on their sequences and were sub-cloned into pUC57.

Construction of level-1 vectors

The level-0 vectors were then used to construct Level-1 vectors: pEC47732: PAtUBI5-GFP-Tmas, pEC47742: PLexA35S-SbMYB75- Thsp18.2, pEC47751: PLexA35S-SbDEL-Tnos, pEC47761: P2×35S-XVE-T35S, according to the protocol:

PCR reaction system of level-1 vectors’ construction
volume / μL
level-1 empty vector (200 ng/μL) 1.0
promoter 1.5
CDS 1.5
terminator 1.5
NEB T4 buffer 1.5
BSA (10×) 1.5
T4 ligase 0.5
BsaI 0.5
ddH2O 10.0
the whole volume 20.0

The reaction ware incubated in a thermocyler for: 37℃ 3 min, 16 ℃ 4 min, 32 cycles;16℃ 15 min;50℃ 5 min,80℃ 5 min. 10 μl of the liagations were used to transform E. coli. Positive clines were confirmed by sequencing.

Construction of level-2 vectors

Level-2 vectors were constructed based on level-1 vectors:

Fig 1. The structure of the NC (BBa_K4399013).
PCR reaction system of level-2 vectors’ construction
items volume / μL
level-2 empty vector (200 ng/μL) 1.0
L1-P1 1.5
L1-P2 1.5
L1-P3 1.5
ELE-X 1.5
NEB T4 buffer 1.5
BSA (10×) 1.5
T4 ligase (NEB) 0.5
BsaI 0.5
ddH2O 9.0
the whole volume 20.0

The reaction ware incubated in a thermocyler for: 37℃ 3 min, 16 ℃ 4 min, 32 cycles; 16℃ 15 min; 50℃ 5 min, 80℃ 5 min. 10 ul of the liagations were used to transform to E. coli. Positive clines were confirmed by sequencing and PCR.

Nucleic acid gel electrophoresis detection results showed that the level-2 vector IA showed one band and its molecular weight were greater than 8000 bp (Fig 2). Monoclonal transformation in plates were obtained by E. coli transformation (Fig 3).PCR detection results showed that SbMYB and SbDEL were integrated in monoclone NC (BBa_K4399013) (Fig 4).

Fig 2. Nucleic acid gel electrophoresis results of plasmid detection for NC (BBa_K4399013).
Fig 3. The plates of E. coli transformants for NC (BBa_K4399013).
Fig 4. Nucleic acid gel electrophoresis results of NC (BBa_K4399013). Line 1-4, PCR results of 4 single colonies of BBa_K4399013 using SbMYB primers; Line 5-8, PCR results of 4 single colonies of BBa_K4399012 using SbMYB primers; Line 9, positive control; Line 10, negative control; Line 11, marker; Line 11-14, PCR results of 4 single colonies of BBa_K4399013 using SbDEL primers; Line 15-18, PCR results of 4 single colonies of BBa_K4399012 using SbDEL primers; Line 19, positive control; Line 20, negative control; Line 21, marker.

Agrobacterium Rhizogenes transformation

The constructed vector NC were successfully transferred into Agrobacterium Rhizogenes A4 by electroporation. The positive transformants containing both SbMYB75 and SbDEL were selected and cultured on solid TY medium plates containing antibiotics of spectinomycin and kanamycin (Fig 5).

Fig 5. The plates of A. Rhizogenes transformants for vector NC.

Explant infection and hairy root emerging induction

Carrot leaves from 4-week-old tissue culture plantlets were used as transformation explants. Carrot leaves were infected by A. Rhizogenes strains containing target genes and co-cultured on the solid MS medium plates containing 50 mM acetosyringone (AS) for 48-72 h at 25°C in the dark and then transferred to MS solid medium containing 400 mg/L cephalexin (cef). 3-5 weeks later after the infection, induced hairy roots emerged from the infection site (Fig 6).

Fig 6. Induced hairy root emerged from the infected leaf explants infected by A. Rhizogenes strains containing vectors NC.

The co-transgenic roots were detected by screening for GFP fluorescence signal.The successful transformants of hairy roots ware marked and transferred on MS plates (without cefotaxime) in dark at 25 °C for further reproduction.

Induction of anthocyanin synthesis

The Carrot hairy roots were cultured in MS liquid medium with 200 mg-L-1 cefotaxime for about 30 days, then 2 μM β-estradiol solution was added in liquid M medium. The results showed that no anthocyanin synthesis was induced in the NC hairy roots on the 1st and 5th day, implying that the negative control for inducible anthocyanin biosynthesis was successfully constructed (Fig 7).


Fig 7. The anthocyanin synthesis induction result of transgenic carrot hairy roots of IA.


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