Difference between revisions of "Part:BBa K1088008"

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.

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

@@ Line 12: / Line 12: @@
 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.
-'''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.
+'''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.