Difference between revisions of "Part:BBa K1431812:Design"

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# Do double enzyme digestion (XbaI & PstI for BBa_K592009, SpeI & PstI for BBa_B0034) and do gel extraction, ligation, transformation, picking up single colonies, LB broth incubation, plasmid extraction and gel electrophoresis verification.
 
# Do double enzyme digestion (XbaI & PstI for BBa_K592009, SpeI & PstI for BBa_B0034) and do gel extraction, ligation, transformation, picking up single colonies, LB broth incubation, plasmid extraction and gel electrophoresis verification.
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# Do double enzyme digestion (XbaI & PstI for Step 1 product, SpeI & PstI for J23100) and do gel extraction, ligation, transformation, picking up single colonies, LB broth incubation, plasmid extraction and gel electrophoresis verification.
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# Do double enzyme digestion (XbaI & PstI for Step 1 product, SpeI & PstI for J23100) and do gel extraction, ligation, transformation, picking up single colonies ('''can select the positive colonies from the colony color'''), LB broth incubation, plasmid extraction and gel electrophoresis verification.
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# Do double enzyme digestion (EcoRI-HF & SpeI for Step 2 product, EcoRI-HF & XbaI for B0015) and do gel extraction, ligation, transformation, picking up single colonies, LB broth incubation, plasmid extraction and gel electrophoresis verification.
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# Do double enzyme digestion (EcoRI-HF & SpeI for Step 2 product, EcoRI-HF & XbaI for B0015) and do gel extraction, ligation, transformation, picking up single colonies ('''can select the positive colonies from the colony color'''), LB broth incubation, plasmid extraction and gel electrophoresis verification.
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# Cryopreserved the rest bacteria broth and send samples for sequencing.
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===Sequencing Results===
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We sent fresh bacteria broth for sequencing using standard Biobricks sequencing primer VF2/VR. The sequencing cooperation we used is Invitrogen Guangzhou filiale.
 +
 
 +
Sequence of sequencing primer we used:<br>
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VF2: tgccacctgacgtctaagaa<br>
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VR: attaccgcctttgagtgagc
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The sequencing result was very interesting: compared with the sequence in Parts Registry, three point mutations were observed, one between NcoI and XbaI enzyme recognition site and two in BBa_J23100 sequence. The following figure shows the detailed information.
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<center>https://static.igem.org/mediawiki/parts/8/8a/SUSTC-Shenzhen-Project-Sequencing_Error-J23100.jpg</center>
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We observed the similar sequencing result in BBa_K1431814 which is also made up of promoter BBa_J23100. We think this sequencing error may explain the unusual observation when comparing the expression with BBa_J23106 because these two mutations weaken the transcriptional strength of BBa_J23100.
  
 
===Source===
 
===Source===
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The following list is the Biobricks we've used and their detailed design information.
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The following list is the Biobricks we've used in construction and their detailed design information.
  
 
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Latest revision as of 01:47, 18 October 2014

amilCP, blue chromoprotein reporter system (Strong Promoter, Strong RBS)


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

This Biobrick is constructed through 3 steps:

  1. Do double enzyme digestion (XbaI & PstI for BBa_K592009, SpeI & PstI for BBa_B0034) and do gel extraction, ligation, transformation, picking up single colonies, LB broth incubation, plasmid extraction and gel electrophoresis verification.
  2. Do double enzyme digestion (XbaI & PstI for Step 1 product, SpeI & PstI for J23100) and do gel extraction, ligation, transformation, picking up single colonies (can select the positive colonies from the colony color), LB broth incubation, plasmid extraction and gel electrophoresis verification.
  3. Do double enzyme digestion (EcoRI-HF & SpeI for Step 2 product, EcoRI-HF & XbaI for B0015) and do gel extraction, ligation, transformation, picking up single colonies (can select the positive colonies from the colony color), LB broth incubation, plasmid extraction and gel electrophoresis verification.
  4. Cryopreserved the rest bacteria broth and send samples for sequencing.

Sequencing Results

We sent fresh bacteria broth for sequencing using standard Biobricks sequencing primer VF2/VR. The sequencing cooperation we used is Invitrogen Guangzhou filiale.

Sequence of sequencing primer we used:
VF2: tgccacctgacgtctaagaa
VR: attaccgcctttgagtgagc

The sequencing result was very interesting: compared with the sequence in Parts Registry, three point mutations were observed, one between NcoI and XbaI enzyme recognition site and two in BBa_J23100 sequence. The following figure shows the detailed information.

SUSTC-Shenzhen-Project-Sequencing_Error-J23100.jpg

We observed the similar sequencing result in BBa_K1431814 which is also made up of promoter BBa_J23100. We think this sequencing error may explain the unusual observation when comparing the expression with BBa_J23106 because these two mutations weaken the transcriptional strength of BBa_J23100.

Source

The following list is the Biobricks we've used in construction and their detailed design information.

Part Type Discription Backbone Location on the plate
BBa_J23100 Promoter Strong Promoter BBa_J61002 (Amp) 17D 2014 Kit Plate 4
BBa_J23106 Promoter Weak Promoter BBa_J61002 (Amp) 17P 2014 Kit Plate 4
BBa_B0031 RBS Weak RBS pSB1A2 1H 2014 Kit Plate 4
BBa_B0034 RBS Strong RBS pSB1A2 1N 2014 Kit Plate 4
BBa_B0015 Terminator Strong terminator (B0010+B0012) pSB1C3 3F 2014 Kit Plate 3
BBa_K592009 Chromoprotein amilCP, blue chromoprotein pSB1C3 19E 2014 Kit Plate 1
BBa_K592011 Chromoprotein cjBlue, green chromoprotein pSB1C3 2I 2014 Kit Plate 4
BBa_K1033916 Chromoprotein amajLime, yellow-green chromoprote pSB1C3 6M 2014 Kit Plate 4

References

  1. Parts Registry Assembly Help
  2. Parts Registry Transformation Guideline