Difference between revisions of "Part:BBa K1088027"

Latest revision as of 18:47, 28 October 2013

Dxs from B. subtilis with lac promoter and lacI (IPTG inducible)

The part consist of the dxs gene derived from B. subtilis under the control of the lac promoter and has a strong RBS.

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

The purpose of the brick is to increase the flow through the MEP pathway upon addition of IPTG.

A similar part (BBa_K1088026) with a GFP fused to Dxs, was used to prove that the part is IPTG inducible.

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 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.

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 2460
Illegal EcoRI site found at 3117
Illegal PstI site found at 2518
Illegal PstI site found at 2964
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 2460
Illegal EcoRI site found at 3117
Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal PstI site found at 2518
Illegal PstI site found at 2964
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 2460
Illegal EcoRI site found at 3117
23
INCOMPATIBLE WITH RFC[23]
Illegal EcoRI site found at 2460
Illegal EcoRI site found at 3117
Illegal PstI site found at 2518
Illegal PstI site found at 2964
25
INCOMPATIBLE WITH RFC[25]
Illegal EcoRI site found at 2460
Illegal EcoRI site found at 3117
Illegal PstI site found at 2518
Illegal PstI site found at 2964
Illegal NgoMIV site found at 2417
Illegal AgeI site found at 2310
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 2259
Illegal SapI.rc site found at 2958

@@ Line 2: / Line 2: @@
 <partinfo>BBa_K1088027 short</partinfo>
-The part consist of the dxs gene derived from B. subtilis under the control of the lac promoter and has a strong RBS.
+The part consist of the ''dxs'' gene derived from ''B. subtilis'' under the control of the lac promoter and has a strong RBS.
-To repress expression from the lac promoter, the lacI gene under a constitutive promoter, with a strong RBS and a efficient terminator is placed counter-clockwise to the reporter fusion. The repression can then be relieved with addition of IPTG, which binds and inhibits the function of LacI.
+To repress expression from the lac promoter, the ''lacI'' gene under a constitutive promoter, with a strong RBS and a efficient terminator is placed upstream to the reporter fusion. The repression can then be relieved with addition of IPTG, which binds and inhibits the function of LacI.
 The purpose of the brick is to increase the flow through the MEP pathway upon addition of IPTG.
@@ Line 14: / Line 14: @@
 FACS results and growth curves of +/-lacI:LVA carrying strains. One triplicate of MG1655 (WT) and two triplicates of MG1655 strains carrying either [https://parts.igem.org/Part:BBa_K1088008 BBa_K1088008] (-''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.
-'''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 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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