Difference between revisions of "Part:BBa K4399012"
Line 15: Line 15:
  
 
== 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 ==

Revision as of 02:19, 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:

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.

Fig 1. Nucleic acid gel electrophoresis results of BBa_K4399012 and 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.