Difference between revisions of "Part:BBa K4960024"
Line 15: Line 15:
  
 
<figure class="figure">
 
<figure class="figure">
−
<p><img src="" alt="" width="800" height="200" /></p>
+
<p><img src="https://static.igem.wiki/teams/4960/wiki/golden-gate.jpg" alt="" width="850" height="600" /></p>
  
 
</figure>
 
</figure>

Revision as of 09:21, 12 October 2023


pvc13 NTD-2*Bsal-pvc13 CTD
A sequence used to introduce the BsaI digestion site.

Profile

Name: pvc13_NTD-2*Bsal-pvc13_CTD
Base Pairs: 1326bp
Origin: Synthetic
Properties: A sequence used to introduce the BsaI digestion site

Usage and Biology

To enable the PVC-based delivery of UCP1 into white adipose tissue, we then decided to equip the tail fiber protein of PVC (PVC13) with an adipose tissue-targeting 9-mer peptide developed by Kolonin et al. in 2004 [1]. To ensure the efficient exposure of the targeting peptide on the tail fiber, we predicted the structure of PVC13 trimers inserted with adipose tissue targeting peptide flanked by different linkers (Figure 1a). We were unable to peruse validating these constructs due to the limited time before the jamboree and the practical difficulties in working with such a huge plasmid (~25kb), while we did make some effort in simplifying the cloning process by designing a new PVC13 part which allows subsequent Golden-gate-based cloning of the targeting sequence into the pPVC plasmid (Figure 4b, see the second part of our Engineering page for more information).

Figure 1. AlphaFold2-guided Design of Adipose Cell-targeting PVC Coat Expressing Plasmids. (a) AlphaFold2 based prediction of engineered PVC tail fiber trimer structure. Structure of adipose-targeting CKGGRAKDC peptide-presenting PVC tail fiber with indicated linkers were shown. (b) Schematic diagram of the newly-designed pvc13 part allowing Golden Gate cloning of targeted sequence into the pPVC plasmid.

Special Design

From many different cloning strategies, we noticed one method named Golden Gate assembly, which achieves hierarchical assembly of DNA parts by utilizing Type IIS restriction enzymes to produce user-specified sticky ends on cut DNA fragments.[2] So we use Golden Gate Assembly to simplify the cloning process by altering the linker on both sides of the CKGGRAKDC to find the most suitable structure. This plasmid can be used as a tool plasmid for a series of subsequent plasmids.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 309
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


References

[1]Kolonin, M. G., Saha, P. K., Chan, L., Pasqualini, R., & Arap, W. (2004). Reversal of obesity by targeted ablation of adipose tissue. Nat Med, 10(6), 625-632. https://doi.org/10.1038/nm1048 [2] Bird, J. E., Marles-Wright, J., & Giachino, A. (2022). A User's Guide to Golden Gate Cloning Methods and Standards. ACS synthetic biology, 11(11), 3551–3563.