Regulatory

Part:BBa_K1088019

Designed by: Patrick Rosendahl Andreassen   Group: iGEM13_SDU-Denmark   (2013-09-17)
Revision as of 12:18, 28 October 2013 by Thigers (Talk | contribs)

LacI device (constitutive promoter, RBS and terminator)

The trancsriptional repressor LacI (BBa_K1088018) under the constitutively active promoter (BBa_J23106) with a RBS (BBa_B0030) and a terminator (BBa_B1002).

This device can be used to overexpress LacI with the purpose of repressing transcription from high-copy plasmids containing the lac promoter (BBa_R0010). The repression can be relieved by allosteric binding of IPTG to LacI, thereby promoting transcription from the lac promoter.

SDU2013_Characterization_LacIPlac_2.2.png

FACS (fluorescence assorted cell sorting) results and growth curves of lacI(N) and lacI:LVA carrying strains. LacI(N) stands for natural LacI, whereas lacI(LVA) contains a LVA tag promoting its fast degradation in the cell and should facilitate a more fine-tuned control. Two triplicates of MG1655 strains carrying either pSB1C3-Pcon-lacI(N)-term-Plac-dxs (B. subtilis)-GFP (BBa_K1088026) (lacI(N)) or pSB1C3-Pcon-lacI:LVA-term-Plac-dxs (B. subtilis)-GFP ([https://parts.igem.org/Part:BBa_K1088009 BBa_K1088009) (lacI:LVA) were grown from OD600 0.005 to approximately 0.2. At this OD the one triplicate of each strain was induced with 1 mM IPTG at time 0 min. FACS measurements were done at times: -30, 0, 30, 60, 90, 120, 150 and 180 min.

A) lacI(lva) and lacI(N) strains grew at the same pace both with and without induction (IPTG). B) Percentage of population above fluorescence threshold. Both lacI(lva) and lacI(N) represses the expression when not induced with IPTG. lacI(lva) reaches a maximum of merely 70-75 % after 150 min, which is both a poor response time and maximum cell percentage fluorescent compared to lacI(N). lacI(N) reaches a maximum just below 100 % at a time between 30 and 60 min after induction. C) Mean GFP fluorescence of the entire population. These results reflect what is seen in B, and clearly indicate that overexpression of natural LacI instead of LacI:LVA is better, when a quick response and high level of expression is required.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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Categories
Parameters
None