Part:BBa_K313017

terminator leakiness assay device

This is a device that aims to measure the leakiness of a terminator BBa_B1006.

If RNA polymerase transcribes the coding region of Cre regardless of the terminator, Cre is expressed from this part.

With our cre recombinase assay device such as BBa_K313016 or BBa_K313009, you would be able to detect the quantity of Cre and assess the leakiness of the terminator.

verification experiment

We tested the function of this Cre recombianse generator.

This part is ligated with double loxP unit (Fig.1A) and transformed into E.coli, JM109 strain.

[Fig.1]

Please note that double loxP unit is inserted between the SpeI site and PstI site of the plasmid containing this part (Double loxP unit was inserted as SpeI and PstI fragment, so SpeI site remains).

Originally the length between SpeI site and PstI site is 1778bp. So, if Cre is not expressed, SP digestion will make 1778bp fragment(Fig.1A). On the other hand, if Cre is expressed, SP digestion will make 726bp fragment(Fig.1B).

We incubated the E.coli for about 20 hours in LB, and then extracted plasmids.Enzyme digestion and sequencing were performed on these samples(Fig.2, Fig.3) and the results proved that the excision occurred between loxP sites.

[fig.2]

A: Plasmid containing double loxP unit (without Cre generator) digested by SpeI and PstI. B: Plasmid containing double loxP unit with this part (Fig.1A) digested by SpeI and PstI M: lambda HindIII marker

[Fig.3] The result of sequence alignment. The upper one is the predicted sequence resulting from the excision by Cre recombinase, and the lower one is the result of sequencing.These two sequences completely match.

We conducted the same experiment on BBa_K313018.

Please see our [http://2010.igem.org/Team:UT-Tokyo/Sudoku_assay_LeakSw Terminator leak] assay page for detail.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 478
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 164
  
• 1000
  COMPATIBLE WITH RFC[1000]