Part:BBa_K3425071

Multi-dummy of TU-DY cloned to pSB3C11

This is a composite part which consists in four instances of TU-DY (BBa_K3425021), each cloned previously to a different pSB1K0#, cloned to pSB3C11 (BBa_K3425001).

Usage and Biology

In order to check whether TU-DY (BBa_K3425021) and MTU-DY (BBa_K3425022) would work as dummy parts, all four pSB1K0#-DY mimicking four TUs were cloned together into pSB3C11. Similarly, all four dummies mimicking MTUs pSB3C1#-DY were cloned into pSB1K02. Sequencing (Figures 11 and 12) revealed that only one out of six clones contains the expected sequence (the last clone of Figure 11). The remaining five clones contain deletions or insertions of various sizes, all of them in the area of the “multi-dummy” sequence.

Figure 11. Detail of the sequencing result of the multi-dummy cloning (TU-DY^4) into pSB3C11. At the upper part, dummy parts, fusion sites and BsaI recognition sequences are annotated. At the bottom, three sequencing reads corresponding to three different clones are shown.

This is probably due to the short and repetitive sequence generated by the dummies, which might have caused polymerase slippage. This phenomenon can occur when there are reiterated sequences, even if they are imperfect [1]. Since the fusion sites at the ends, next to the BsaI and SapI recognition sequences, are present in all the clones it is likely that the cloning was successful and the polymerase slippage happened afterwards, during the growth of the transformed cells.

Figure 12. Detail of the sequencing result of the multi-dummy cloning (MTU-DY^4) into pSB1K02. At the upper part, dummy parts, fusion sites and BsaI recognition sequences are annotated. At the bottom, three sequencing reads corresponding to three different clones are shown.

Dummy parts are only supposed to be fillers that allow the cloning reaction to ligate properly when you have less than four parts to assemble. In this sense, this dummy sequence works as intended. However, it is not ideal to have unpredictable mutations occurring systematically.

A simple solution would be not cloning the dummy sequences next to each other. There might be a situation where this is not possible, so in that case it might be good to use longer dummy parts that are all different from each other. These parts were designed in silico and correspond to part BBa_K3425023 to BBa_K3425030.

Sequencing results of this part can be found in the Sequence Analysis section and the trace files are in the collection page as well as our Parts page.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 14
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 14
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 14
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 14
  
• 1000
  COMPATIBLE WITH RFC[1000]