Difference between revisions of "Part:BBa K1741011"

Revision as of 08:34, 27 September 2015

sfGFP under T7 promoter

This part imitates the most popular IPTG/lactose dependent T7 viral RNA polymerase driven expression systems. To express another sequence of interest sfGFP could be substituted using Gibson or CPEC assembly method. Host strain of choice expressing T7 RNA polymerase is necessary, not only DE3.

Results

We compared our promoters to T7 promoter on minimal M9 medium with added glucose.

The sfGFP fluorescence [RFU] was measured using Tecan fluorometer.

The expression rate under T7 promoter is high compared to other tested promoters even though there is no inducer (lactose) present in the medium (so it has a tendency to leak). Our promoters do not exhibit any significant expression. Note that this comparison is simplified, because the transcription from T7 promoter is performed by strong viral polymerase while the transcription from our other promoters is performed by slower cellular RNA polymerase.

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal XbaI site found at 48
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
INCOMPATIBLE WITH RFC[23]
Illegal XbaI site found at 48
25
INCOMPATIBLE WITH RFC[25]
Illegal XbaI site found at 48
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI.rc site found at 131

@@ Line 2: / Line 2: @@
 <partinfo>BBa_K1741011 short</partinfo>
-This part imitates the most popular IPTG/lactose dependent T7 driven expression systems. To express sfGFP or another sequence with which one can substitute sfGFP ORF using Gibson or CPEC assembly, host strain of choice expressing T7 RNA polymerase is necessary, not only DE3.
+This part imitates the most popular IPTG/lactose dependent T7 viral RNA polymerase driven expression systems. To express another sequence of interest sfGFP could be substituted using Gibson or CPEC assembly method. Host strain of choice expressing T7 RNA polymerase is necessary, not only DE3.
 ===Results===
@@ Line 12: / Line 12: @@
 The sfGFP fluorescence [RFU] was measured using Tecan fluorometer.
-As you can see, there is high expression of T7 promoter however there is no inducer (lactose) present in the medium (so it has a tendency to leak). Our promoters don't show any significant expression. Please note that this comparison is simplified, because the transcription from T7 promoter is performed by strong viral polymerase while the transcription from our other promoters is performed by slower cellular RNA polymerase.
+The expression rate under T7 promoter is high compared to other tested promoters even though there is no inducer (lactose) present in the medium (so it has a tendency to leak). Our promoters do not exhibit any significant expression.
+Note that this comparison is simplified, because the transcription from T7 promoter is performed by strong viral polymerase while the transcription from our other promoters is performed by slower cellular RNA polymerase.