Part:BBa_K2996012
pLac upstream of repL
RepL is a P1 lytic replication element that can act on mini-F origin to promote plasmid replication. In this composite part, pLac is put upstream of it, which enables IPTG to regulate the expression of repL. We transfered plasmid containing this part into E.coli BL21(DE3). When induced by IPTG, the copy of plasmid increased in a great deal. The sharp increase of plasmid copy number served as a trigger of signal input in the biostorage system of our project.
Functional Parameters
When induced by 0.5mM IPTG, we measured the copy number change through a time course using qPCR and the result showed obvious increase in the plasmid copy number. The maximum increase was about 2000 times of copy more than without induction which appeared at around 4.5h of induction. A sequence from genome served as internal control.
Protocol
(1)Transfer ptrig(with pLac driven repL) into E.coli BL21(DE3).
(2)Inoculate 1% overnight bacterial culture in Erlenmeyer flask.
(3)Incubate for 2h to reach early exponential stage (OD600 is 0.2-0.3).
(4)Add IPTG to final concentration of 0.5 mM.
(5)Take 500μL of the culture every 60min after 3h induction.
(6)Heat at 100℃ for 10min.
(7)Store at -20℃ for at least 20min.
(8)Centrifuge at 6500rpm for 1min.
(9)Remove the supernatant into a clean 1.5mL EP tube.
(10)Store at -20℃.
(11)Add 3ul as qPCR template and run qPCR.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 328
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1049
- 1000COMPATIBLE WITH RFC[1000]
| None |