Difference between revisions of "Part:BBa K1088008"
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The purpose of the part was to test the expression profile of ''dxs'' from the lac promter. BBa_K1088011 is a similar part that does not contain the linker-GFP part.  
 
The purpose of the part was to test the expression profile of ''dxs'' from the lac promter. BBa_K1088011 is a similar part that does not contain the linker-GFP part.  
  
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Fluorescence activated cell sorting (FACS) was used to examine the expression profile the reporter fusion. The part was examined in the K-12 MG1655 (natural levels of LacI) and KG22 (overexpression of LacI from chromosome) strains. The experiment proved that overexpression of LacI is required for repression of the lac promoter (and therefore also control of the regulation). BBa_K1088026 is a similar part which overexpresses LacI from the part itself.
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Fluorecense activated cell sorting was used to measure protein levels, and a similar device (BBa_K1088009) to this containing yet another a device overexpressing lacI:LVA as well, was used for comparison.
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https://static.igem.org/mediawiki/2013/3/3c/SDU2013_Part_BBa_K1088020.png
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FACS results and growth curves of +/-lacI:LVA carrying strains. One triplicate of MG1655 (WT) and two triplicates of MG1655 strains carrying either this part (-lacI:LVA) or BBa_K1088009 (+lacI:LVA) were grown from OD<sub>600</sub> 0.005 to approximately 0.2. At this OD the MG1655 triplicate and one triplicate of each strain carrying constructs were induced with 1 mM IPTG at time 0 min. FACS measurements were done at times: -30, 0, 30, 60, 90, 120, and 150 min.
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A) Growth curve shows that WT grows slightly faster than strains bearing plasmids. B) Per cent of population above fluorescence threshold. None of the WT cells were fluorescent, almost all of the -lacI:LVA cells were constitutively fluorescent, and only cells overexpressing LacI:LVA weren’t fluorescent when not induced. Upon induction increasingly per cent of +lacI:LVA became fluorescent and reaches a maximum of 70-75 per cent after 90 min. C) Mean GFP fluorescence of entire population. The -lacI:LVA cells became increasingly more fluorescent over time, both with and without induction. Though, the induced cells were slightly more fluorescent, which is probably because of the relief of repression from LacI naturally present in the cells. For the +lacI:LVA cells the results from B is reflected.
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In conclusion this part is constitutively active when LacI isn't overexpressed in the cell.  
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 23:49, 3 October 2013

B. subtilis dxs-GFP fusion (lac promoter without lac inhibitor: IPTG uninducible)

This part consist of the dxs gene derived from B. subtilis fused to GFP at the translational level with a 10 AA linker between the proteins. The reporter fusion is under the control of the lac promoter and has a strong RBS.

The purpose of the part was to test the expression profile of dxs from the lac promter. BBa_K1088011 is a similar part that does not contain the linker-GFP part.

Fluorecense activated cell sorting was used to measure protein levels, and a similar device (BBa_K1088009) to this containing yet another a device overexpressing lacI:LVA as well, was used for comparison.

SDU2013_Part_BBa_K1088020.png

FACS results and growth curves of +/-lacI:LVA carrying strains. One triplicate of MG1655 (WT) and two triplicates of MG1655 strains carrying either this part (-lacI:LVA) or BBa_K1088009 (+lacI:LVA) were grown from OD600 0.005 to approximately 0.2. At this OD the MG1655 triplicate and one triplicate of each strain carrying constructs were induced with 1 mM IPTG at time 0 min. FACS measurements were done at times: -30, 0, 30, 60, 90, 120, and 150 min. A) Growth curve shows that WT grows slightly faster than strains bearing plasmids. B) Per cent of population above fluorescence threshold. None of the WT cells were fluorescent, almost all of the -lacI:LVA cells were constitutively fluorescent, and only cells overexpressing LacI:LVA weren’t fluorescent when not induced. Upon induction increasingly per cent of +lacI:LVA became fluorescent and reaches a maximum of 70-75 per cent after 90 min. C) Mean GFP fluorescence of entire population. The -lacI:LVA cells became increasingly more fluorescent over time, both with and without induction. Though, the induced cells were slightly more fluorescent, which is probably because of the relief of repression from LacI naturally present in the cells. For the +lacI:LVA cells the results from B is reflected.

In conclusion this part is constitutively active when LacI isn't overexpressed in the cell.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1279
    Illegal EcoRI site found at 1936
    Illegal PstI site found at 1337
    Illegal PstI site found at 1783
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1279
    Illegal EcoRI site found at 1936
    Illegal PstI site found at 1337
    Illegal PstI site found at 1783
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1279
    Illegal EcoRI site found at 1936
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1279
    Illegal EcoRI site found at 1936
    Illegal PstI site found at 1337
    Illegal PstI site found at 1783
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1279
    Illegal EcoRI site found at 1936
    Illegal PstI site found at 1337
    Illegal PstI site found at 1783
    Illegal NgoMIV site found at 1236
    Illegal AgeI site found at 1129
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1078
    Illegal BsaI.rc site found at 2792
    Illegal SapI.rc site found at 1777