Difference between revisions of "Part:BBa K5439003:Design"
 
Line 1: Line 1:
−
With the DNA fragments purified from an agarose gel, we performed ligation at a molar ratio of 1:5 for vector and insert, as shown in <b>Figure 3</b>. The total vector concentration was 100 nanograms, whereas the reaction volume was 20 µL. Next, <b>Table 2</b> displays the protocol followed for the reaction.
+
__NOTOC__
 +
<partinfo>BBa_K5439003short</partinfo>
 +
<div style="text-align:justify;">
 +
<partinfo>BBa_K5439003 SequenceAndFeatures</partinfo>
  
−
{| class="wikitable" style="margin:auto; text-align:center; length: 80%"
+
===Design Notes===
−
|+ Table 2. DNA ligation conditions.
+
A gly-gly-ser spacer, and a polyhistidine-tag were added before stop codon for protein purification. NcoI and XhoI restriction sites were added in 5' and 3' ends for protein overexpression in pBAD/Myc-His plasmids. There are no scars between each protein. Crystal structures of RifMo from Nocardia farcinica have been reported, providing valuable insights into its molecular architecture and potential mechanisms of action Liu <i>et al.</i> (2016).  
−
|-
+
 
−
!Reactive !! Quantity
+
 
−
|-
+
===Source===
−
| style="text-align:center;" style="width: 80%;" | T4 DNA Ligase Buffer (10X) || 2 µL
+
 
−
|-
+
The original sequence was reported by Liu <i>et al.</i> (2016). Sequence can be obtained through GenBank (CP000946). The sequence for BBa_K1159302 was reported by  Poëa-Guyon <i>et al.</i> (2013). The sequence for BBa_K1907000 was reported by Nagai <i>et al.</i> (2002).
−
| style="text-align:center;" style="width: 80%;" | Vector DNA || 100 ng
+
 
−
|--
+
===References===
−
| style="text-align:center;" style="width: 80%;" | Insert DNA || 773.5 ng
+
 
−
|-
+
[1]. Liu, L.-K., Abdelwahab, H., Martin Del Campo, J. S., Mehra-Chaudhary, R., Sobrado, P., & Tanner, J. J. (2016). The Structure of the Antibiotic Deactivating, N-hydroxylating Rifampicin Monooxygenase. Journal of Biological Chemistry, 291(41), 21553–21562. https://doi.org/10.1074/jbc.m116.745315
−
| style="text-align:center;" style="width: 80%;" | Nuclease-free water || To 20 µL
+
 
−
|-
+
[2].  Poëa-Guyon, S., Pasquier, H., Mérola, F., Morel, N., & Erard, M. (2013). The enhanced cyan fluorescent protein: a sensitive pH sensor for fluorescence lifetime imaging. <i>Analytical and bioanalytical chemistry, 405</i>(12), 3983–3987. https://doi.org/10.1007/s00216-013-6860-y
−
| style="text-align:center;" style="width: 80%;" | T4 DNA Ligase || 1.5 µL
+
 
−
|}
+
[3]. Nagai, T., Ibata, K., Park, E. S., Kubota, M., Mikoshiba, K., & Miyawaki, A. (2002). A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. <i>Nature Biotechnology, 20</i>(1), 87–90. doi:10.1038/nbt0102-87

Latest revision as of 07:24, 2 October 2024

No part name specified with partinfo tag.


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 1947
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 2178
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 2833

Design Notes

A gly-gly-ser spacer, and a polyhistidine-tag were added before stop codon for protein purification. NcoI and XhoI restriction sites were added in 5' and 3' ends for protein overexpression in pBAD/Myc-His plasmids. There are no scars between each protein. Crystal structures of RifMo from Nocardia farcinica have been reported, providing valuable insights into its molecular architecture and potential mechanisms of action Liu et al. (2016).


Source

The original sequence was reported by Liu et al. (2016). Sequence can be obtained through GenBank (CP000946). The sequence for BBa_K1159302 was reported by Poëa-Guyon et al. (2013). The sequence for BBa_K1907000 was reported by Nagai et al. (2002).

References

[1]. Liu, L.-K., Abdelwahab, H., Martin Del Campo, J. S., Mehra-Chaudhary, R., Sobrado, P., & Tanner, J. J. (2016). The Structure of the Antibiotic Deactivating, N-hydroxylating Rifampicin Monooxygenase. Journal of Biological Chemistry, 291(41), 21553–21562. https://doi.org/10.1074/jbc.m116.745315

[2]. Poëa-Guyon, S., Pasquier, H., Mérola, F., Morel, N., & Erard, M. (2013). The enhanced cyan fluorescent protein: a sensitive pH sensor for fluorescence lifetime imaging. Analytical and bioanalytical chemistry, 405(12), 3983–3987. https://doi.org/10.1007/s00216-013-6860-y

[3]. Nagai, T., Ibata, K., Park, E. S., Kubota, M., Mikoshiba, K., & Miyawaki, A. (2002). A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nature Biotechnology, 20(1), 87–90. doi:10.1038/nbt0102-87