Regulatory

Part:BBa_K2605001

Designed by: Kaitlin Schaaf   Group: iGEM18_Calgary   (2018-10-09)


CMV with 5' SalI restriction site

This is an improvement of Part BBa_K747096

Overview

The Cytomegalovirus (CMV) promoter is a strong constitutive promoter commonly used in mammalian expression systems The addition of the SalI restriction site allows for the promoter to be used within our Multiple-Cloning Site flanked by FRT sites Part BBa_K2605002 system. The SalI restirction site has the sequence GTCGAC.


Experimental Characterization

Restriction enzyme digest was used to confirm the addition of the SalI restriction site to the CMV promoter. pSB1C3-BBa_K2605001 and pSB1C3-BBa_K747096 were digested with either NotI (to confirm part size) or SalI/SpeI (to confirm the presence of the SalI restriction site). As expected, NotI cut both plasmids and produced a roughly 600kb insert while SalI/SpeI only produced a 600kb insert from our improved part, pSB1C3-BBa_K2605001. Since pSB1C3-BBa_K747096 only contains a SpeI site, the plasmid was linearized and the insert band was not produced. The results of this restriction enzyme digest can be seen below (figure 1).

Figure 1. Digest confirmations of [A] pSB1C3-BBa_K2605001 and [B]pSB1C3-BBa_K747096 . The plasmid was double digested with SalII and SpeI or single digested with NotI then run on a 1.5% agarose gel at 100 V for 35 minutes. The molecular ladder (L) is visible on the far left and the expected band sizes, obtained from Benchling Virtual Digest, are visible in the diagram on the right. Undigested plasmid (U) was used as a control.


To verify that our improved part could still function as a promoter, both parts were ligated to a red fluorescent protein (RFP) reporter gene optimized for bacteria Part:BBa_J06504, which was found in the 2018 iGEM distribution kit. Although CMV promoters are normally used for mammalian expression, it also functions as a promoter in E. coli (Lewin et al., 2005), therefore it could be used to express the RFP reporter gene that was fused to it.

Digest-confirmed CMV-RFP constructs and RFP alone (pSB1C3-BBa_J06504, negative control) were transformed into chemically competent E. coli DH5-alpha cells. Successful transformants were plated and imaged after 48h, at which time RFP production could be seen in both CMV-RFP constructs, while the negative control remained colourless as RFP could not be produced without a CMV promoter (figure 2, figure 3).


Figure 2. Streak plates of E. coli DH5-alpha containing TOP: pSB1C3-BBa_J06504 (RFP) fused to BBa_K747096 (CMV), RIGHT: pSB1C3-BBa_J06504 (RFP) fused to BBa_K2605001 (CMV) LEFT: negative control, pSB1C3-BBa_J06504 (RFP).


Figure 3. Streak plates of E. coli DH5-alpha containing TOP: pSB1C3-BBa_J06504 (RFP) fused to BBa_K747096 (CMV), BOTTOM: pSB1C3-BBa_J06504 (RFP) fused to BBa_K2605001 (CMV)

References

Lewin, A., Mayer, M., Chusainow, J., Jacob, D., Appel, B. (2005). Viral promoters can initiate expression of toxin genes introduced into Escherichia coli. Biomed Central Biotechnology, 5(19). doi: 10.1186/1472-6750-5-19.


Sequence and Features

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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