Part:BBa_G00000

BioBrick cloning site prefix

Used as a basic part in BioBrick vectors.

Report by Kyoto2023

This year the iGEM Kyoto team used the simplified homologous recombination cloning method (referred to as XE cloning) for the creation of almost all plasmids, as described in the article "Quick and affordable DNA cloning by reconstitution of Seamless Ligation Cloning Extract using defined factors [1]" was used for the creation of almost all plasmids. This method is similar to the commercially available Gibson Assembly [2] and InFusion HD cloning [3], or the SLiCE method [4]. The details of this process can be found in the paper.

When creating some plasmids, we used the JUMP plasmid as a template, amplified the vector by PCR, and attempted to assemble it with artificially synthesized DNA fragments and PCR products by XE cloning. In this case, to be able to use the same primer for multiple types of JUMP plasmids and to avoid leaving scars, we decided to use primers that allow assembly of the BioBrick prefix and the BioBrick suffix as homologous arms respectively.

However, the bands obtained very frequently from the electrophoresis results of colony-direct PCR were thought to have been assembled by the vector itself without inserts.

Fig.1 Results of colony direct PCR

Sixteen DH5α-transformed pGM018 colonies were randomly selected for colony-direct PCR. If the inserts had been inserted correctly, a band of 2599 bp would have been observed, but all bands appeared in the vicinity of 500 bp, indicating that the vectors had cycled themselves.

The sequence of the plasmid that was supposed to be assembled by the vector itself was confirmed by Sanger sequencing, and the sequence result is shown in Fig.2.

Fig.2 The sequence obtained as a result of Sanger sequencing

Based on our sequencing results, we concluded that the vectors were assembled mainly because they share the same 8 bases of NotI in prefix and suffix, as shown in Fig.3.

Fig.3 The hypothesized mechanism of assembly in the vector

There is an 8-base complementary region in the NotI region. There are other complementary regions, and 11 bp of the 16 bp overlap region is complementary. Interestingly, 6 bases outside of NotI on the suffix side and 2 bp outside of NotI on the prefix side were shaved by E. coli and replaced by complementary bases.

To solve this problem, the overlap region was set up so as not to include NotI, and primers were designed for this purpose.

Fig.4 A view of primer design, with an overlap region on the insert side rather than the BioBrick prefix to prevent overlaps near NotI

As a result, The expected bands were obtained with high frequency.

Fig5 Results of colony direct PCR

Bands of expected length were observed except in lane 3. The bands were smeared, probably because the plasmid was made to express nuclease (nucA), which may have degraded some DNA.

From the above, we found that the unintended vector-to-vector assembly was caused by homology in the NotI portion, and can be prevented by designing the overlap region to not contain NotI. In the following experiments, primers were designed based on these results.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal prefix found in sequence at 1
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1
  Illegal NotI site found at 7
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found in sequence at 1
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found in sequence at 1
  Illegal XbaI site found at 16
  
• 1000
  COMPATIBLE WITH RFC[1000]

Reference

[1] Liu, A. Y., Koga, H., Goya, C., & Kitabatake, M. (2023). Quick and affordable DNA cloning by reconstitution of Seamless Ligation Cloning Extract using defined factors. Genes to Cells: Devoted to Molecular & Cellular Mechanisms, 28(8), 553–562.

[2] Gibson, D. G., Young, L., Chuang, R.-Y., Venter, J. C., Hutchison, C. A., 3rd, & Smith, H. O. (2009). Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods, 6(5), 343–345.

[3] Zhu, B., Cai, G., Hall, E. O., & Freeman, G. J. (2007). In-fusion assembly: seamless engineering of multidomain fusion proteins, modular vectors, and mutations. BioTechniques, 43(3), 354–359.

[4] Zhang, Y., Werling, U., & Edelmann, W. (2012). SLiCE: a novel bacterial cell extract-based DNA cloning method. Nucleic Acids Research, 40(8), e55.