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.
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. Thus, we confirmed proper generation of Cre recombinase from this parts, and its correct operation.
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
The result of sequence alignment. 3A : 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.
3B : The upper one is original sequence before the excision and the lower one is result of sequencing. It is clear that about 1052bp between loxP sites was excised.
Although one terminator exists upstream of Cre generator and the strain used in this experiment doesn't have T7 RNA polymerase, the excision did occur. This result indicates the possibility that transcription occurred from unpredicted region and terminator did not stop this transcription and Cre was expressed.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 478
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 164
- 1000COMPATIBLE WITH RFC[1000]
| None |