Difference between revisions of "Part:BBa K1088009"
 
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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.
 
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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To repress expression from the ''lac'' promoter, the ''lacI:LVA'' (gene under a constitutive promoter, with a strong RBS and an efficient terminator) was placed counter-clockwise to the reporter fusion. The repression can be relieved with addition of IPTG, which binds and inhibits the function of LacI:LVA.  
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To repress expression from the ''lac'' promoter, the ''lacI:LVA'' (gene under a constitutive promoter, with a strong RBS and an efficient terminator) was placed upstream to the reporter fusion. The repression can be relieved with addition of IPTG, which binds and inhibits the function of LacI:LVA.  
  
 
This brick was build to assay the expression profile before and after induction of a similar device ([https://parts.igem.org/Part:BBa_K1088013 BBa_K1088013]) which lacks the linker and GFP.
 
This brick was build to assay the expression profile before and after induction of a similar device ([https://parts.igem.org/Part:BBa_K1088013 BBa_K1088013]) which lacks the linker and GFP.
  
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Fluorescense activated cell sorting (FACS) was used to measure protein expression, and a similar device (BBa_K1088009) without the part that overexpresses lacI:LVA 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_K1088008 BBa_K1088008]) without the part that overexpresses 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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'''A)''' Growth curve shows that WT cells grow 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 cells 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 is reflected.  
 
'''A)''' Growth curve shows that WT cells grow 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 cells 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 is reflected.  
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https://static.igem.org/mediawiki/2013/7/73/SDU2013_Expression_FACS3.png
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'''Explanatory text:'''
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FACS results and growth curves of <i>lacI(N)</i> and <i>lacI:LVA</i> carrying strains. Dublicates of MG1655 carrying either pSB1C3-Pcon-<i>lacI(N)</i>-term-Plac-<i>dxs (B. subtilis)</i>-GFP ([https://parts.igem.org/Part:BBa_K1088026 BBa_K1088026]) (<i>lacI(N)</i>) or pSB1C3-Pcon-<i>lacI:LVA</i>-term-Plac-<i>dxs (B. subtilis)</i>-GFP (this part) (<i>lacI(lva)</i>) were grown from OD<sub>600</sub>=0.005 to approximately OD<sub>600</sub>=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. <b> A)</b> <i>lacI(lva)</i> and <i>lacI(N)</i> strains grew at the same pace <b> B)</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 <i>lacI(lva)</i> 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 <i>lacI(N)</i> induced with IPTG concentrations from 0.125-0.5mM become fluorescent within 180 min, and cultures with higher IPTG concentrations reach this point sooner.
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Latest revision as of 21:18, 29 October 2013

B. subtilis dxs-GFP protein fusion (lac promoter with LVA-tagged lac inhibitor (LacI:LVA) - IPTG ind

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.

To repress expression from the lac promoter, the lacI:LVA (gene under a constitutive promoter, with a strong RBS and an efficient terminator) was placed upstream to the reporter fusion. The repression can be relieved with addition of IPTG, which binds and inhibits the function of LacI:LVA.

This brick was build to assay the expression profile before and after induction of a similar device (BBa_K1088013) which lacks the linker and GFP.

Fluorescense activated cell sorting (FACS) was used to measure protein expression, and a similar device (BBa_K1088008) without the part that overexpresses 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 BBa_K1088008(-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 cells grow 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 cells 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 is reflected.


SDU2013_Expression_FACS3.png Explanatory text: 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 (this part) (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.

Sequence and Features

Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 2505
    Illegal EcoRI site found at 3162
    Illegal PstI site found at 2563
    Illegal PstI site found at 3009
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 2505
    Illegal EcoRI site found at 3162
    Illegal NheI site found at 7
    Illegal NheI site found at 30
    Illegal PstI site found at 2563
    Illegal PstI site found at 3009
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 2505
    Illegal EcoRI site found at 3162
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 2505
    Illegal EcoRI site found at 3162
    Illegal PstI site found at 2563
    Illegal PstI site found at 3009
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 2505
    Illegal EcoRI site found at 3162
    Illegal PstI site found at 2563
    Illegal PstI site found at 3009
    Illegal NgoMIV site found at 2462
    Illegal AgeI site found at 2355
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 2304
    Illegal BsaI.rc site found at 4018
    Illegal SapI.rc site found at 3003