Difference between revisions of "Part:BBa K4399012"
 
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It is an inducible anthocyanin biosynthesis system, which contains a constitutive GFP expression box, an inducible SbMYB75 expression box, an inducible SbDEL expression box and a constitutive XVE expression box.  
 
It is an inducible anthocyanin biosynthesis system, which contains a constitutive GFP expression box, an inducible SbMYB75 expression box, an inducible SbDEL expression box and a constitutive XVE expression box.  
 +
 +
[[File:BBa K4399012-Fig1b.png|600px|thumb|center| '''Fig 1. The structure of the IA (BBa_K4399012).''']]
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
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== Construction of level-0 vectors ==
 
== Construction of level-0 vectors ==
−
The DNA elements (golden gate compatible, promoters: P<sub>AtUBI5</sub>, P<sub>LexA35S</sub>, P<sub>2×35S</sub>; CDS of Genes: GFP, SbMYB75, SbDEL, XVE; Terminators: T<sub>mas</sub>, T<sub>hsp18.2</sub>, T<sub>nos</sub>, T<sub>35S</sub>) were synthesized by Gensript based on their sequences and were sub-cloned into pUC57. \times
+
The DNA elements (golden gate compatible, promoters: P<sub>AtUBI5</sub>, P<sub>LexA35S</sub>, P<sub>2×35S</sub>; CDS of Genes: GFP, SbMYB75, SbDEL, XVE; Terminators: T<sub>mas</sub>, T<sub>hsp18.2</sub>, T<sub>nos</sub>, T<sub>35S</sub>) were synthesized by Gensript based on their sequences and were sub-cloned into pUC57.
  
 
== Construction of level-1 vectors ==
 
== Construction of level-1 vectors ==
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|+ PCR reaction system of level-1 vectors’ construction
 
|+ PCR reaction system of level-1 vectors’ construction
 
|-
 
|-
−
!   !! volume / μL
+
! items !! volume / μL
 
|-
 
|-
 
| level-1 empty vector (200 ng/μL) || 1.0
 
| level-1 empty vector (200 ng/μL) || 1.0
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|+ PCR reaction system of level-2 vectors’ construction
 
|+ PCR reaction system of level-2 vectors’ construction
 
|-
 
|-
−
!   !! IA !! NC
+
! items !! volume / μL
 
|-
 
|-
−
!  !! volume / μL !! volume / μL
+
| level-2 empty vector (200 ng/μL) || 1.0
 
|-
 
|-
−
| level-2 empty vector (200 ng/μL) || 1.0 || 1.0
+
| L1-P1 || 1.5
 
|-
 
|-
−
| L1-P1 || 1.5 || 1.5
+
| L1-P2 || 1.5
 
|-
 
|-
−
| L1-P2 || 1.5 || 1.5
+
| L1-P3 || 1.5
 
|-
 
|-
−
| L1-P3 || 1.5 || 1.5
+
| L1-P4 || 1.5
 
|-
 
|-
−
| L1-P4 || 1.5 || -
+
| ELE-X || 1.5
 
|-
 
|-
−
| ELE-X || 1.5 || 1.5
+
| NEB T4 buffer || 1.5
 
|-
 
|-
−
| NEB T4 buffer || 1.5 || 1.5
+
| BSA (10×) || 1.5
 
|-
 
|-
−
| BSA (10×) || 1.5 || 1.5
+
| T4 ligase (NEB) || 0.5
 
|-
 
|-
−
| T4 ligase (NEB) || 0.5 || 0.5
+
| BsaI || 0.5
 
|-
 
|-
−
| BsaI || 0.5 || 0.5
+
| ddH<sub>2</sub>O || 7.5
 
|-
 
|-
−
| ddH<sub>2</sub>O || 7.5 || 9
+
| the whole volume || 20.0
−
|-
+
−
| the whole volume || 20.0 || 10.0
+
 
|}
 
|}
  
−
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 E. coli. Positive clines were confirmed by sequencing and PCR.
+
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 IA (BBa_K4399012) ('''Fig 4''').
 +
 
 +
[[File:BBa K4399012-Fig2.png|300px|thumb|center| '''Fig 2. Nucleic acid gel electrophoresis results of plasmid detection for IA (BBa_K4399012).''']]
 +
 
 +
[[File:BBa K4399012-Fig3.png|300px|thumb|center| '''Fig 3. The plates of ''E. coli'' transformants for IA (BBa_K4399012).''']]
  
−
[[File:BBa K4399012-Fig1.png|600px|thumb|center| '''Fig 1. Nucleic acid gel electrophoresis results of BBa_K4399012 and BBa_K4399013.'''
+
[[File:BBa K4399012-Fig4.png|600px|thumb|center| '''Fig 4. Nucleic acid gel electrophoresis results of IA (BBa_K4399012).'''
  
  
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Line 21, marker.]]
 
Line 21, marker.]]
 +
 +
== ''Agrobacterium Rhizogenes'' transformation ==
 +
The constructed vector IA 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''').
 +
 +
[[File:BBa K4399012-Fig5.png|300px|thumb|center| '''Fig 5. The plates of ''A. Rhizogenes'' transformants for vector IA.''']]
 +
 +
== 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''').
 +
 +
[[File:BBa K4399012-Fig6.png|300px|thumb|center| '''Fig 6. Induced hairy root emerged from the infected leaf explants infected by ''A. Rhizogenes'' strains containing vectors IA.''']]
 +
 +
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 anthocyanin synthesis was successfully induced in the IA hairy roots on the 1st and 5th day, implying that the inducible anthocyanin biosynthesis system was successfully constructed ('''Fig 7''').
 +
 +
 +
[[File:BBa K4399012-Fig7.png|600px|thumb|center| '''Fig 7. The anthocyanin synthesis induction result of transgenic carrot hairy roots of IA.''']]
 +
 +
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K4399012 parameters</partinfo>
 
<partinfo>BBa_K4399012 parameters</partinfo>
 
<!-- -->
 
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Latest revision as of 04:54, 8 October 2022


The inducible anthocyanin biosynthesis system

It is an inducible anthocyanin biosynthesis system, which contains a constitutive GFP expression box, an inducible SbMYB75 expression box, an inducible SbDEL expression box and a constitutive XVE expression box.

Fig 1. The structure of the IA (BBa_K4399012).

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 XbaI site found at 8415
    Illegal PstI site found at 2759
    Illegal PstI site found at 4179
    Illegal PstI site found at 4264
    Illegal PstI site found at 8599
    Illegal PstI site found at 8770
  • 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
    Illegal PstI site found at 8599
    Illegal PstI site found at 8770
  • 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 BglII site found at 6994
    Illegal BglII site found at 7321
    Illegal BglII site found at 8549
    Illegal BamHI site found at 3547
    Illegal BamHI site found at 3849
    Illegal BamHI site found at 5168
    Illegal BamHI site found at 5237
    Illegal BamHI site found at 8118
    Illegal XhoI site found at 7525
  • 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 XbaI site found at 8415
    Illegal PstI site found at 2759
    Illegal PstI site found at 4179
    Illegal PstI site found at 4264
    Illegal PstI site found at 8599
    Illegal PstI site found at 8770
  • 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 XbaI site found at 8415
    Illegal PstI site found at 2759
    Illegal PstI site found at 4179
    Illegal PstI site found at 4264
    Illegal PstI site found at 8599
    Illegal PstI site found at 8770
  • 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
items 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:

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
L1-P4 1.5
ELE-X 1.5
NEB T4 buffer 1.5
BSA (10×) 1.5
T4 ligase (NEB) 0.5
BsaI 0.5
ddH2O 7.5
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 IA (BBa_K4399012) (Fig 4).

Fig 2. Nucleic acid gel electrophoresis results of plasmid detection for IA (BBa_K4399012).
Fig 3. The plates of E. coli transformants for IA (BBa_K4399012).
Fig 4. Nucleic acid gel electrophoresis results of IA (BBa_K4399012). 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 IA 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 IA.

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 IA.

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 anthocyanin synthesis was successfully induced in the IA hairy roots on the 1st and 5th day, implying that the inducible anthocyanin biosynthesis system was successfully constructed (Fig 7).


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