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Part:BBa_K1384000:Experience

Designed by: Anuved Verma   Group: iGEM14_UCLA   (2014-10-08)


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Applications of BBa_K1384000

2015 UCLA iGEM

We attempted to use BBa_K1384000 to perform Iterative Capped Assembly, but were unsuccessful. We believe that this is due to non-specific binding of the designed sticky end. Please see our user review below for more details.

User Reviews

UNIQbf9f7bd0d9485741-partinfo-00000000-QINU UNIQbf9f7bd0d9485741-partinfo-00000001-QINU

2015 UCLA iGEM

  • This part does not work as intended. We attempted to use this part to perform ICA, but the other pieces required to perform ICA were not present as part of this collection. In addition, the sticky ends present in this Biobrick are non-specific, and will form undesirable concatemers.
  • We believe the non-specificity of the old pieces is due to a high GC content in the sticky end. In addition, there is only one base pair difference between each of the sticky ends. This may have also contributed to non-specificity.
  • We performed a ligation test using AB, BC, CA sticky ends by ligating two of the monomer pieces together. If the sticky ends work as intended, there would be one ligation band corresponding to the 2-mer product.
Fig.1 Ligation test using monomer pairs. A represents AB, B is BC, and C is CA. The presence of laddering bands indicates non-specific binding, showing that this Biobrick is unsuitable for use in ICA.
  • Over the course of summer 2015, we were able to develop a new [http://2015.igem.org/Team:UCLA/Notebook/Spider_Silk_Genetics#ICA_Parts_Collection collection of biobricks]. We were able to successfully use this collection to perform ICA.
  • Our new sticky ends had a reduced GC content as well as a two base pair difference between the sticky ends.
  • We show below a similar ligation test performed using our parts to examine the ligation behavior of our newly designed sticky ends.
Fig. 2 Ligation test of AB, BC, CA. Lane 1 is A+B, Lane 2 is B+C, Lane 3 is C+A.
  • In each lane, there is only one ligation product present. This indicates that our redesigned sticky ends work as intended, and serve as an improvement over the older design.