Difference between revisions of "Part:BBa K4399012"
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== Construction of level-2 vectors ==
 
== Construction of level-2 vectors ==
 
Level-2 vectors were constructed based on level-1 vectors:
 
Level-2 vectors were constructed based on level-1 vectors:
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[[File:BBa K4399012-Fig1b.png|600px|thumb|center| '''Fig 1. The structure of the IA (BBa_K4399012).'''
  
 
{| class="wikitable" style="margin:auto"
 
{| class="wikitable" style="margin:auto"
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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 E. coli. Positive clines were confirmed by sequencing and PCR.
  
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[[File:BBa K4399012-Fig4.png|600px|thumb|center| '''Fig 4. Nucleic acid gel electrophoresis results of BBa_K4399012 and BBa_K4399013.'''
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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''').
 +
 
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[[File:BBa K4399012-Fig2.png|600px|thumb|center| '''Fig 2. Nucleic acid gel electrophoresis results of plasmid detection for IA (BBa_K4399012).'''
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[[File:BBa K4399012-Fig3.png|600px|thumb|center| '''Fig 2. The plates of E. coli transformants for IA (BBa_K4399012).'''
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[[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.]]
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== ''Agrobacterium Rhizogenes'' transformation ==
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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 (Figure 5).
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[[File:BBa K4399012-Fig5.png|600px|thumb|center| '''Fig 5. The plates of A. Rhizogenes transformants for vector IA.'''
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<!-- 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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Revision as of 04:03, 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.

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
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:

[[File:BBa K4399012-Fig1b.png|600px|thumb|center| Fig 1. The structure of the IA (BBa_K4399012).

PCR reaction system of level-2 vectors’ construction
IA NC
volume / μL volume / μL
level-2 empty vector (200 ng/μL) 1.0 1.0
L1-P1 1.5 1.5
L1-P2 1.5 1.5
L1-P3 1.5 1.5
L1-P4 1.5 -
ELE-X 1.5 1.5
NEB T4 buffer 1.5 1.5
BSA (10×) 1.5 1.5
T4 ligase (NEB) 0.5 0.5
BsaI 0.5 0.5
ddH2O 7.5 9
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.

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|600px|thumb|center| Fig 2. Nucleic acid gel electrophoresis results of plasmid detection for IA (BBa_K4399012).

[[File:BBa K4399012-Fig3.png|600px|thumb|center| Fig 2. 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 (Figure 5).

[[File:BBa K4399012-Fig5.png|600px|thumb|center| Fig 5. The plates of A. Rhizogenes transformants for vector IA.