Difference between revisions of "Part:BBa K2940014"
Jakub Chromy (Talk | contribs) |
Jakub Chromy (Talk | contribs) |
||
| Line 3: | Line 3: | ||
<partinfo>BBa_K2940014 short</partinfo> | <partinfo>BBa_K2940014 short</partinfo> | ||
| − | This part encodes the biosensor genetic circuit comprising the toluene-specific transcription factor XylR and the sfGFP reporter gene. This part is a modification of the BBa_K2023015 part originally developed by Ionis Paris iGEM team 2016 [1]. The part was meant to serve as the starting biosensor circuit platform for the subsequent directed evolution of XylR in order to develop a biosensor for the detection of aromatic amines. | + | This part encodes the biosensor genetic circuit comprising the toluene-specific transcription factor XylR and the sfGFP reporter gene. This part is a modification of the BBa_K2023015 part originally developed by Ionis Paris iGEM team 2016 [1]. The part was meant to serve as the starting biosensor circuit platform for the subsequent directed evolution of XylR in order to develop a biosensor for the detection of aromatic amines. |
| + | |||
===Usage and Biology=== | ===Usage and Biology=== | ||
BTEX (Benzene, Toluene, Ethylbenzene, Xylene)-like aromatic compounds detection. | BTEX (Benzene, Toluene, Ethylbenzene, Xylene)-like aromatic compounds detection. | ||
| + | |||
===Characterization=== | ===Characterization=== | ||
| Line 13: | Line 15: | ||
The E. coli TOP10 cells transformed with XylR:sfGFP_pSB1C3 biosensor plasmid exhibited strong fluorescence even when uninduced. This strong background fluorescence was most presumably the result of sfGFP leaky expression and made cells apparently not sensitive to the induction when grown on plain and 10 mg/L toluene-containing LB agar plates (Figure 1). | The E. coli TOP10 cells transformed with XylR:sfGFP_pSB1C3 biosensor plasmid exhibited strong fluorescence even when uninduced. This strong background fluorescence was most presumably the result of sfGFP leaky expression and made cells apparently not sensitive to the induction when grown on plain and 10 mg/L toluene-containing LB agar plates (Figure 1). | ||
| − | [[Image:T--Edinburgh OG--Figure1 Jakub.png|400px| | + | [[Image:T--Edinburgh OG--Figure1 Jakub.png|400px|Figure 1]] |
| + | |||
| + | |||
| + | Indeed, there were no significant differences in fluorescence responses between uninduced and 10 mg/L toluene-induced cells when accurately measured by a plate reader (Figure 2A). | ||
| + | |||
| + | [[Image:T--Edinburgh OG--Figure2 Jakub.png|400px|Figure 2]] | ||
| + | |||
| + | The situation was not improved even by inverting the direction of the XylR sensing module and sfGFP reporter module, so they face opposite directions and their transcription is really independent (Figure 2B), nor when using the low-copy pSB3C5 and pSB4C5 plasmid backbones (Figure 2C). | ||
| + | |||
| + | |||
| + | ===Conclusion=== | ||
| + | |||
| + | Our results suggest, that the codon-optimized sfGFP variant might not be a suitable reporter in certain biosensor settings, as any leaky expression can create very strong fluorescence background signal obscuring the actual signal. | ||
| + | |||
| + | |||
<!-- --> | <!-- --> | ||
Revision as of 22:27, 20 October 2019
XylR coding device-sfGFP_optimism coding device with Pu
This part encodes the biosensor genetic circuit comprising the toluene-specific transcription factor XylR and the sfGFP reporter gene. This part is a modification of the BBa_K2023015 part originally developed by Ionis Paris iGEM team 2016 [1]. The part was meant to serve as the starting biosensor circuit platform for the subsequent directed evolution of XylR in order to develop a biosensor for the detection of aromatic amines.
Usage and Biology
BTEX (Benzene, Toluene, Ethylbenzene, Xylene)-like aromatic compounds detection.
Characterization
The E. coli TOP10 cells transformed with XylR:sfGFP_pSB1C3 biosensor plasmid exhibited strong fluorescence even when uninduced. This strong background fluorescence was most presumably the result of sfGFP leaky expression and made cells apparently not sensitive to the induction when grown on plain and 10 mg/L toluene-containing LB agar plates (Figure 1).
Indeed, there were no significant differences in fluorescence responses between uninduced and 10 mg/L toluene-induced cells when accurately measured by a plate reader (Figure 2A).
The situation was not improved even by inverting the direction of the XylR sensing module and sfGFP reporter module, so they face opposite directions and their transcription is really independent (Figure 2B), nor when using the low-copy pSB3C5 and pSB4C5 plasmid backbones (Figure 2C).
Conclusion
Our results suggest, that the codon-optimized sfGFP variant might not be a suitable reporter in certain biosensor settings, as any leaky expression can create very strong fluorescence background signal obscuring the actual signal.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 2446
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 2101
Illegal XhoI site found at 3018 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 329
- 1000COMPATIBLE WITH RFC[1000]