Difference between revisions of "Part:BBa K1088018"

Revision as of 20:43, 29 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.

Explanatory text: 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 (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.

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.

To test its qualities compared to LacI:LVA further, another FACS was performed. The experimental setup was similar to the previous. Dublicates of MG1655 strains carrying either pSB1C3-Pcon-lacI(N)-term-Plac-dxs (B. subtilis)-GFP or pSB1C3-Pcon-lacI:LVA-term-Plac-dxs (B. subtilis)-GFP were induced with IPTG concentrations rangingfrom 0.05-0.5mM (Fig. 3). After 180 min upon induction, the strains expressing LacI:LVA with the IPTG concentrations ranging from 0.05-0.5mM reached approximately 25-30 % cells above the fluorescent threshold. Comparing this observation to the strains overexpressing the natural LacI, even the least induced (0.05mM) reached a higher percentage of cells above the fluorescent threshold after 180 min. All of the cells in the culture overexpressing natural LacI induced with 0.125-0.5mM IPTG are above the fluorescent threshold within 180 min. In general, the response time is faster for higher concentrations of IPTG.

<a class="popupImg alignCenter" href="" title="Figure 3 - FACS results and growth curves of lacI(N) and lacI:LVA carrying strains. Dublicates of MG1655 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 OD₆₀₀=0.2. At this OD, the one triplicate of each strain was induced with 0.05, 0.125, 0.25, or 0.5 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 B) Percentage of population above fluorescenct threshold. Upon induction of lacI(lva) with different concentrations of IPTG, the percentage of bacteria becoming fluorescent slowly increases. After approximately 180 min, 25-30 % of lacI(lva) induced bacteria in the range from 0.05-0.5 mM IPTG are above the fluorescenct threshold. After 180 min, there is a slightly higher percentage of bacteria above the fluorescenct threshold than for the lacI(lva) strain induced with 10-fold higher IPTG concentration. All lacI(N) induced with IPTG concentrations from 0.125-0.5mM become fluorescent within 180 min, and cultures with higher IPTG concentrations reach this point sooner. These results support what was seen in figure 2"> </a>

Sequence and Features