Difference between revisions of "Part:BBa K731040"

Revision as of 12:13, 20 September 2012

Inducible araC-pBAD promoter regulating a sfGFP-tagged M256I CysE

A sfGFP tagged M256I CysE inducible by arabinose. This part was subcloned and characterized in pSB3C5.

Usage and Biology

FIGURE 1. CysE-sfGFP expression with different concentrations of arabinose, after 4 hours of induction.
Test was conducted in NEB10beta strain, with part BBa_K731240, but in pSB3C5. Cells were grown in LB brothuntil OD_600= 0.3 then centrifuged and resuspended in equal amount of minimal MOPS A medium (with 60mM glycerol as carbon source) and induced at OD_600 = 0.6 with 5mM arabinose concentration. Higher CysE-sfGFP expression is observed as the arabinose concentration increases. However, MOPS medium seem to negatively influence induction, delaying promoter response to higher arabinose concentration. No difference in fluorescence intensity between not induced and control NEB10beta cells was observed.

FIGURE 2. CysE-sfGFP expression with different concentrations of arabinose in presence or absence of glucose.
This chart shows the difference in expression levels when using glucose or glycerol as carbon source. Expression levels in MOPS 30mM glucose with 5mM arabinose are very similar to levels in MOPS 60mM glycerol and 0.1mM arabinose.

FIGURE 3. CysE-sfGFP expression over time, after induction.
Test was conducted in DH10beta strain, with part BBa_K731250, but in pSB3C5. Cells were grown in LB brothuntil OD_600= 0.3 then centrifuged and resuspended in equal amount of minimal MOPS medium and induced at OD_600 = 0.6 with 5mM arabinose concentration. MOPS A is 60mM glycerol as carbon source, MOPS B is 30mM glucose: in the chart, data relative to MOPS A are shown in orange, the ones relative MOPS B are in blue. In absence of glucose, increasing linear expression over time of GFP is observed, while in MOPS B, in presence of glucose, expression levels are much lower.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]

12
COMPATIBLE WITH RFC[12]

21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 1144
Illegal BamHI site found at 2045

23
COMPATIBLE WITH RFC[23]

25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 979
Illegal AgeI site found at 1909

1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI site found at 961
Illegal SapI.rc site found at 2075

@@ Line 4: / Line 4: @@
 A sfGFP tagged M256I CysE inducible by arabinose. This part was subcloned and characterized in pSB3C5.
-<!-- Add more about the biology of this part here
 ===Usage and Biology===
+<div style="text-align:center">[[Image:1040_1.jpg]]</div>
+<html><p style="width:600px; margin-left:150px; margin-bottom:60px;
+text-align:justify "><em><strong>FIGURE 1. CysE-sfGFP expression with different concentrations of arabinose, after 4 hours of induction.</strong><br/>Test was conducted in NEB10beta strain, with part BBa_K731240, but in pSB3C5. Cells were grown in LB brothuntil OD_600= 0.3 then centrifuged and resuspended in equal amount of minimal MOPS A medium (with 60mM glycerol as carbon source) and induced at OD_600 = 0.6 with 5mM arabinose concentration. Higher CysE-sfGFP expression is observed as the arabinose concentration increases. However, MOPS medium seem to negatively influence induction, delaying promoter response to higher arabinose concentration. No difference in fluorescence intensity between not induced and control NEB10beta cells was observed.</p></html>
+<div style="text-align:center">[[Image:1040_2.jpg]]</div>
+<html><p style="width:600px; margin-left:150px; margin-bottom:60px;
+text-align:justify "><em><strong>FIGURE 2. CysE-sfGFP expression with different concentrations of arabinose in presence or absence of glucose.</strong><br/>This chart shows the difference in expression levels when using glucose or glycerol as carbon source. Expression levels in MOPS 30mM glucose with 5mM arabinose are very similar to levels in MOPS 60mM glycerol and 0.1mM arabinose.</p></html>
+<div style="text-align:center">[[Image:1040_3.jpg]]</div>
+<html><p style="width:600px; margin-left:150px; margin-bottom:60px;
+text-align:justify "><em><strong>FIGURE 3. CysE-sfGFP expression over time, after induction.</strong><br/>Test was conducted in DH10beta strain, with part BBa_K731250, but in pSB3C5. Cells were grown in LB brothuntil OD_600= 0.3 then centrifuged and resuspended in equal amount of minimal MOPS medium and induced at OD_600 = 0.6 with 5mM arabinose concentration. MOPS A is 60mM glycerol as carbon source, MOPS B is 30mM glucose: in the chart, data relative to MOPS A are shown in orange, the ones relative MOPS B are in blue. In absence of glucose, increasing linear expression over time of GFP is observed, while in MOPS B, in presence of glucose, expression levels are much lower. </p></html>
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