Difference between revisions of "Part:BBa K1088009"

Revision as of 12:46, 26 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 counter-clockwise 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_K1088009) without the part that overexpresses lacI:LVA was used for comparison.

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.

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

@@ Line 2: / Line 2: @@
 <partinfo>BBa_K1088009 short</partinfo>
-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.
-To repress expression from the lac promoter, the ''lacI:LVA'' (gene under a constitutive promoter, with a strong RBS and a 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.
+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.
-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.
+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.
-Fluorecense activated cell sorting was used to measure protein levels, and a similar device (BBa_K1088009) to this without the part that overexpresses lacI:LVA was used for comparison.
+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.
 https://static.igem.org/mediawiki/2013/3/3c/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 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.
-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.
+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.