Difference between revisions of "Part:BBa K1088018"

Revision as of 18:17, 26 October 2013

LacI repressor from E. coli

Coding sequence for the LacI protein. LacI functions by binding to the lac promoter BBa_R0010 and inhibits transcription. The inducer IPTG binds LacI and inhibits its function, and thus promotes 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. 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 (BBa_K1088009) (lacI:LVA) were grown from OD₆₀₀ 0.005 to approximately 0.2. At this OD the one triplicate of each strain were 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. 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.

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
INCOMPATIBLE WITH RFC[10]
Unknown
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Unknown
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

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 https://static.igem.org/mediawiki/2013/5/5d/SDU2013_Characterization_LacIPlac_2.2.png
-FACS results and growth curves of lacI(N) and lacI:LVA carrying strains. 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 (BBa_K1088009) (lacI:LVA) were grown from OD<sub>600</sub> 0.005 to approximately 0.2. At this OD the one triplicate of each strain were 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.
+FACS (fluorescence assorted cell sorting) results and growth curves of lacI(N) and lacI:LVA carrying strains. 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 (BBa_K1088009) (lacI:LVA) were grown from OD<sub>600</sub> 0.005 to approximately 0.2. At this OD the one triplicate of each strain were 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.
 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.
-A)  lacI(lva) and lacI(N) strains grew at the same pace both with and without induction (IPTG). B) Percent 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.
+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.
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