Part:BBa_K3288034
pCP25-RBS(Dawn) - mScarlet
We selected four strong promoters and three strong RBSs to find a strong constitutive expression system. Twelve regulatory parts were constructed using a combination of four strong promoters and three strong RBSs. MScarlet was used as a reporter for monitoring their expression intensity.
Usage and Biology
From an existing pool of strong promoters and rbs, we selected and experimented with the expression of the new combination of units. For the promoters, we used CP25, CP44, J23119 and OXB20 (promoter of pSF-OXB20, a commercial constitutive expression vector), all commonly used in iGEM. For the rbs, we used CC2 clone from cdog library, one already referenced in iGem, NO29 based on the following research paper, and the one included in the pDawn vector. We used mScarlet as a reporter (Excitation;569 nm, Emission;594 nm). Constitutive expression unit plasmids were transformed in E. coli BL21 and overnight cultured in LB(+Amp). After well grown E. coli were transferred to agar plate, they were incubated for 4 hours at 37 ℃. Images were obtained with fluorescence stereo microscope equipped a CCD camera. Among the 12 constitutive expression units (CEU), pCEU03, 06, 09 and 12 that includes RBS originated from pDawn had the highest expression of mScarlet. The expression of pCEU03, 06, and 09 were too high, limiting the growth of E. coli. Therefore, we used PCEU 12 for the rest of the experiment.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1
Illegal XhoI site found at 809 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 318
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