Difference between revisions of "Part:BBa K1088008"
 
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<partinfo>BBa_K1088008 short</partinfo>
 
<partinfo>BBa_K1088008 short</partinfo>
  
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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.
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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.
  
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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.  
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The purpose of the part was to test the expression profile of ''dxs'' from the ''lac'' promter. [https://parts.igem.org/Part:BBa_K1088011 BBa_K1088011] is a similar part that does not contain the linker-GFP part.  
  
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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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Fluorescense activated cell sorting (FACS) was used to measure protein expression, and a similar device ([https://parts.igem.org/Part:BBa_K1088009 BBa_K1088009]) to this containing yet another device overexpressing ''lacI:LVA'', was used for comparison.   
  
 
https://static.igem.org/mediawiki/2013/3/3c/SDU2013_Part_BBa_K1088020.png
 
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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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 [https://parts.igem.org/Part:BBa_K1088009 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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'''A)''' Growth curve shows that the WT grows slightly faster than strains carrying plasmids. '''B)''' Percent 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 increasing percent of +''lacI:LVA'' carrying bacteria became fluorescent and reached a maximum of 70-75 percent after 90 min. '''C)''' Mean GFP fluorescence of the entire population. The -''lacI:LVA'' cells became increasingly more fluorescent over time, both with and without induction. 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 are reflected.  
  
 
In conclusion this part is constitutively active when LacI isn't overexpressed in the cell.  
 
In conclusion this part is constitutively active when LacI isn't overexpressed in the cell.  

Latest revision as of 18:13, 28 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.

Fluorescense activated cell sorting (FACS) was used to measure protein expression, and a similar device (BBa_K1088009) to this containing yet another device overexpressing lacI:LVA, 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 the WT grows slightly faster than strains carrying plasmids. B) Percent 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 increasing percent of +lacI:LVA carrying bacteria became fluorescent and reached a maximum of 70-75 percent after 90 min. C) Mean GFP fluorescence of the entire population. The -lacI:LVA cells became increasingly more fluorescent over time, both with and without induction. 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 are 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