Difference between revisions of "Part:BBa K2014002"
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===Usage and Biology=== | ===Usage and Biology=== | ||
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+ | The construct <b>pxylS-E1_5’UTR->sfGFP</b> is composed of XylA part of <i>Eschericha coli</i> K-12 double sided xylose promoter in which we exchanged the native downstream 5’UTR with a <b>E1_5’UTR</b> (Fig. 1). <b>E1_5’UTR</b> contains an additional ribosome binding site from gene 10 of bacteriophage T7. The new synthetic promoter controls sfGFP expression. The fluorescent protein sfGFP is a marker of gene expression and protein synthesis/accumulation. Protein expression from all compared xylose responsive promoters was induced in rich media with 0.4% D-xylose. | ||
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+ | {|align="center" | ||
+ | |-valign="top" | ||
+ | | colspan = 2 | [[Image:BBa K2014002-1.png|thumb|500px|center|<font size="2"><b>Fig. 1 Synthetic evolution of <i>E. coli</i> xylose induced promoters in our lab.</b> pxylS-E1_5’UTR (XylS-E1) promoter contains only XylA part of <i>E. coli</i> double sided xylose operon promoter, since XylF part appeared to be very weak, and E1_5’UTR with the additional ribosome binding site from gene 10 of bacteriophage T7. </font>]] | ||
+ | |} | ||
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+ | <b>pxylS-E1_5’UTR is most likely the strongest available version of a xylose induced promoter in <i>E. coli</i>.</b><br> | ||
+ | <b>pxylS-E1_5’UTR (XylS-E1)</b> is the strongest among all xylose promoters provided so far to the iGEM community by our team UAM_Poznan (Fig. 2). XylS-E1 ensures aproximately 2-3-fold higher expression than its wild-type version (BBa_K1741007) and slightly lower than T7 promoter from pET systems (Fig. 3). | ||
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+ | {|align="center" | ||
+ | |-valign="top" | ||
+ | | colspan = 2 | [[Image:BBa K2014002-2.png|thumb|500px|center|<font size="2"><b>Fig. 2 Comparison between pxylS-E1_5’UTR (XylS-E1) and our previous xylose responsive promoters.</b> <i>E. coli</i> DH5α transformed with appropriate constructs containing sfGFP were cultured for 6h in LB medium containing 0.4% xylose.</font>]] | ||
+ | |} | ||
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+ | {|align="center" | ||
+ | |-valign="top" | ||
+ | | colspan = 2 | [[Image:BBa K2014002-3.png|thumb|500px|center|<font size="2"><b>Fig. 3 Comparison between pxylS-E1_5’UTR (XylS-E1) and its wild-type version (XylWT)- left panel. Comparison between pxylS-E_15’UTR (XylS-E1) and T7 promoter- right panel.</b> <br> | ||
+ | <i>E. coli</i> DH5α (transformed with XylWT, XylSE1) or BL21-DE3 (transformed with T7-sfGFP) cells were cultured for 6h in LB medium supplemented with 0.4% xylose or lactose respectively. | ||
+ | </font>]] | ||
+ | |} | ||
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+ | <b>pxylS-E1_5’UTR</b> activity was tested in <i>E. coli</i> DH5α grown in three different media (1xLB, 2xLB, SB-PKB) containing 0.4% xylose (Fig. 4). pxylS-E1_5’UTR was compared to previous xylose promoter versions: XylWT (BBa_K1741007), XylA1 (BBa_K1741008), XylS (BBa_K1741009) and to negative control – XylS promoter driving lysozyme gene expression to have identical growth conditions with no fluorescent protein production. The promoter pxylS-E1_5’UTR provided the highest protein expression in all media, at least 2-times higher than its wild-type version (Fig.4, Fig. 5). | ||
+ | |||
+ | |||
+ | {|align="center" | ||
+ | |-valign="top" | ||
+ | | colspan = 2 | [[Image:BBa K2014002-4.png|thumb|600px|center|<font size="2"><b>Fig. 4 Comparison between pxylS-E1_5’UTR (XylS-E1) and our previous xylose promoter versions in <i>E. coli</i> cultured in 1xLB, 2xLB, SB-PKB.</b> <i>E. coli</i> DH5α with appropriate constructs containing sfGFP were cultured for 6h, after induction with D-xylose (0.4% final conc.). The efficiency of promoters was compared based on relative fluorescence intensity of produced sfGFP. OD<sub>600</sub> shows that the growth rate of <i>E. coli</i> in all compared cultures is very similar (bacterial cultures were diluted 10-times).</font>]] | ||
+ | |} | ||
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+ | {|align="center" | ||
+ | |-valign="top" | ||
+ | | colspan = 2 | [[Image:BBa K2014002-5.png|thumb|500px|center|<font size="2"><b>Fig. 5 Photograph illustrating sfGFP fluorescence, which expression was controlled by different versions of xylose inducible promoters;</b> <i>E. coli</i> DH5α were cultured for 6h in SB-PKB medium, with 0.4% xylose. Our newest promoter pxylS-E1_5’UTR (XylS-E1) is the strongest one.</font>]] | ||
+ | |} | ||
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+ | <u>Xylose promoters- legend:</u><br> | ||
+ | xylF-xylA - formerly called XylWT <b>([https://parts.igem.org/Part:BBa_K1741007 BBa_K1741007])</b><br> | ||
+ | xylF-xylA-proD5'UTR - formerly called XylA1 <b>([https://parts.igem.org/Part:BBa_K1741008 BBa_K1741008])</b><br> | ||
+ | xylA-proD5'UTR - formerly called XylS <b>([https://parts.igem.org/Part:BBa_K1741009 BBa_K1741009])</b><br> | ||
+ | xylA-M5'UTR – formerly called XylS-UTR <b>([https://parts.igem.org/Part:BBa_K2014004 BBa_K2014004])</b><br><br> | ||
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+ | References:<br> | ||
+ | 1. Olins PO, Rangwala SH.; A novel sequence element derived from bacteriophage T7 mRNA acts as an enhancer of translation of the lacZ gene in Escherichia coli. J Biol Chem. 1989 Oct 15;264(29):16973-6.<br> | ||
+ | 2. Davis J.H., Rubin A.J., Sauer R.T.; Design, construction and characterization of a set of insulated bacterial promoters. Nucleic Acids Research, 2011, Vol. 39, No. 3 1131–1141<br> | ||
+ | 3. Song S., Park C.; Organization and Regulation of the D-Xylose Operons in Escherichia coli K-12: XylR Acts as a Transcriptional Activator. Journal of Bacteriology, Nov. 1997, p. 7025–7032<br> | ||
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Latest revision as of 23:53, 21 October 2016
pxylS-E1_5'UTR->sfGFP
Usage and Biology
The construct pxylS-E1_5’UTR->sfGFP is composed of XylA part of Eschericha coli K-12 double sided xylose promoter in which we exchanged the native downstream 5’UTR with a E1_5’UTR (Fig. 1). E1_5’UTR contains an additional ribosome binding site from gene 10 of bacteriophage T7. The new synthetic promoter controls sfGFP expression. The fluorescent protein sfGFP is a marker of gene expression and protein synthesis/accumulation. Protein expression from all compared xylose responsive promoters was induced in rich media with 0.4% D-xylose.
pxylS-E1_5’UTR is most likely the strongest available version of a xylose induced promoter in E. coli.
pxylS-E1_5’UTR (XylS-E1) is the strongest among all xylose promoters provided so far to the iGEM community by our team UAM_Poznan (Fig. 2). XylS-E1 ensures aproximately 2-3-fold higher expression than its wild-type version (BBa_K1741007) and slightly lower than T7 promoter from pET systems (Fig. 3).
pxylS-E1_5’UTR activity was tested in E. coli DH5α grown in three different media (1xLB, 2xLB, SB-PKB) containing 0.4% xylose (Fig. 4). pxylS-E1_5’UTR was compared to previous xylose promoter versions: XylWT (BBa_K1741007), XylA1 (BBa_K1741008), XylS (BBa_K1741009) and to negative control – XylS promoter driving lysozyme gene expression to have identical growth conditions with no fluorescent protein production. The promoter pxylS-E1_5’UTR provided the highest protein expression in all media, at least 2-times higher than its wild-type version (Fig.4, Fig. 5).
Xylose promoters- legend:
xylF-xylA - formerly called XylWT (BBa_K1741007)
xylF-xylA-proD5'UTR - formerly called XylA1 (BBa_K1741008)
xylA-proD5'UTR - formerly called XylS (BBa_K1741009)
xylA-M5'UTR – formerly called XylS-UTR (BBa_K2014004)
References:
1. Olins PO, Rangwala SH.; A novel sequence element derived from bacteriophage T7 mRNA acts as an enhancer of translation of the lacZ gene in Escherichia coli. J Biol Chem. 1989 Oct 15;264(29):16973-6.
2. Davis J.H., Rubin A.J., Sauer R.T.; Design, construction and characterization of a set of insulated bacterial promoters. Nucleic Acids Research, 2011, Vol. 39, No. 3 1131–1141
3. Song S., Park C.; Organization and Regulation of the D-Xylose Operons in Escherichia coli K-12: XylR Acts as a Transcriptional Activator. Journal of Bacteriology, Nov. 1997, p. 7025–7032
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 174