Difference between revisions of "Part:BBa K1741009"

Latest revision as of 00:48, 22 October 2016

sfGFP under shortened promoter xylA1 with improved 5'UTR

Design

Legend:

Genes involved in catabolism of xylose in E.coli are organized into two transcription units which are driven by two promoters - XylA and XylF. In order to increase the expression of a protein under control of our XylA1 promoter we added the 5'UTR of the proD promoter[BBa_K1741014] to the XylA of XylWT. In some experiments it seems to be slightly stronger than the original XylA promoter from part BBa_K1741007. The next step was to shorten the promoter by removing its XylF part while keeping the modified 5'UTR (from proD) behind XylA. Finally we tested the reversed XylF part of the XylWT promoter with sfGFP as the reporter gene. XylS is the strongest xylose-induced promoter. Xylose induced promoter XylA1 is weaker than arabinose and rhamnose promoters.

Results

Legend:

XylWT (XylA) [BBa_K1741007]

XylA1 [BBa_K1741008]

XylS [BBa_K1741009]

XylF [BBa_K1741010]

All our xylose promoters are induced by xylose and repressed by glucose. We also observed slight induction by arabinose. Short version of XylA1 promoter with known functional elements is stronger than two other versions (BBa_K1741007, BBa_K1741008). We observed that XylA1 and XylF had a lower expression than XylWT. XylS is the strongest xylose-induced promoter.

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

We also checked the tightness of our promoters.

All of our promoters are induced only by their respective sugars with a small exception of xylose promoters being slightly induced by arabinose and arabinose promoters being slightly induced by xylose. All of our promoters are repressed by glucose.

Contribution

Group: UAM_Poznan 2016
Author: Daria Niewiadomska

The promoter pxylS-M5'UTR (with a lab name: XylS-UTR ), tested in a biobrick pxylS-M5'UTR->sfGFP (BBa_K2014004) is a modified xylose-induced promoter/5’UTR controlling sfGFP protein expression. To make it we exchanged the 5’UTR sequence of xylA-proD5'UTR in a biobrick (BBa_K1741009) to a synthetic, unstructured M5’UTR (picture B) derived from the Mel2 promoter (BBa_K1741004), which we provided to iGEM Registry in 2015.

If you are interested in the pathway of our modifications please click here-> MODIFICATIONS OF XYLOSE INDUCED PROMOTERS

Biobricks used in description:

xylF-xylA - briefly called XylWT (BBa_K1741007)
xylF-xylA-proD5'UTR - briefly called XylA1 (BBa_K1741008)
xylA-proD5'UTR - briefly called XylS (BBa_K1741009)
xylS-M5'UTR - briefly called XylS-UTR (BBa_K2014004)

Figure 1 Synthetic evolution of E. coli xylose induced promoters in our lab up to xylS-UTR. The promoter pxylS-M5'UTR (XylS-UTR) contains only xylA part of E. coli double sided xylose operon promoter, since xylF part appeared to be very weak. The synthetic, unstructured M5’UTR containing a strong, well-positioned RBS slightly enhances the responsiveness of xylA promoter to xylose. Despite that the promoters are stronger, they are still tight (Figure 2.).

Figure 2 Comparison of xylose promoters tightness during overnight time course cultures of E. coli DH5α in 1xLB medium without xylose. Bacteria were transformed with constructs: XylWT (xylF-xylA->sfGFP; BBa_K1741007), XylS (xylA-proD5'UTR; BBa_K1741009), XylS-UTR (xylS-M5'UTR; BBa_K2014004) and XylS-lysozyme (the construct containing non fluorescent protein- lysozyme, under XylS protein), which was used as our background.

Figure 3
A) The most likely secondary structure that can be folded from the 5’UTR of XylWT promoter. B) The most likely secondary structures of M5’UTR from Mel2 and XylS promoters (BBa_K1741004). The sequence of M5’UTR is designed to minimize the likelihood of secondary structure formation. Structures shown above, were generated by RNAFold (http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi) under the same parameters. RBS sequences are underlined with a green line.

Figure 4
A) Xylose inducibility of four xyl promoters/UTRs during 6h time course cultures of E. coli DH5α in LB medium containing 0.4% xylose. The efficiency of the improved promoter pxylS-M5'UTR (BBa_K2014004) is slightly higher, than all other versions of xylose responsive promoters we tested, and its induction seems to be faster (see 2h time-point). B) The growth rate of all constructs compared: Xyl-WT (BBa_K1741007), XylA1 (BBa_K1741008), XylS (BBa_K1741009) or XylS-UTR (BBa_K2014004) is very similar.

Sequence and Features

Assembly Compatibility:

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COMPATIBLE WITH RFC[10]
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COMPATIBLE WITH RFC[12]
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COMPATIBLE WITH RFC[21]
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COMPATIBLE WITH RFC[23]
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COMPATIBLE WITH RFC[25]
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INCOMPATIBLE WITH RFC[1000]
Illegal SapI.rc site found at 185

@@ Line 2: / Line 2: @@
 <partinfo>BBa_K1741009 short</partinfo>
-==Design==
+===Design===
 Legend:
@@ Line 15: / Line 15: @@
-Genes involved in catabolism of xylose in E.coli are organized into two transcription units which are driven by two promoters - XylA and XylF. In order to increase the expression of a protein under our XylA1 promoter we have modified the 5'UTR of the XylWT promoter so it was similar to that of proD [BBa_K1741014], a strong constitutive promoter. In some experiments it seems to be slightly stronger than original XylA promoter from part BBa_K1741007.
+Genes involved in catabolism of xylose in ''E.coli'' are organized into two transcription units which are driven by two promoters - XylA and XylF. In order to increase the expression of a protein under control of our XylA1 promoter we added the 5'UTR of the proD promoter[BBa_K1741014] to the XylA of XylWT. In some experiments it seems to be slightly stronger than the original XylA promoter from part BBa_K1741007. The next step was to shorten the promoter by removing its XylF part while keeping the modified 5'UTR (from proD) behind XylA. Finally we tested the reversed XylF part of the XylWT promoter with sfGFP as the reporter gene.
-The next step was to shorten the promoter by removing its XylF part while keeping the modified UTR. Finally we tested the XylF promoter upstream of sfGFP. XylS is the strongest xylose-induced promoter.
+XylS is the strongest xylose-induced promoter. Xylose induced promoter XylA1 is weaker than arabinose and rhamnose promoters.
-Xylose induced promoter XylA1 is weaker than arabinose and rhamnose promoters.
 ===Results===
@@ Line 41: / Line 40: @@
 All our xylose promoters are induced by xylose and repressed by glucose. We also observed slight induction by arabinose. Short version of XylA1 promoter with known functional elements is stronger than two other versions (BBa_K1741007, BBa_K1741008). We observed that XylA1 and XylF had a lower expression than XylWT. XylS is the strongest xylose-induced promoter.
-The sfGFP fluorescence [RFU] was measured using Tecan fluoremeter.
+The sfGFP fluorescence [RFU] was measured using Tecan fluorometer.
@@ Line 49: / Line 48: @@
 https://static.igem.org/mediawiki/parts/4/43/Teamuampoznanpromoterscomparisongrpah.png
+===Contribution===
+<b>Group:</b> UAM_Poznan 2016 <br />
+<b>Author:</b> Daria Niewiadomska <br />
+The promoter pxylS-M5'UTR (with a lab name: XylS-UTR ), tested in a biobrick pxylS-M5'UTR->sfGFP <b>[https://parts.igem.org/Part:BBa_K2014004 (BBa_K2014004)]</b> is a modified xylose-induced promoter/5’UTR controlling sfGFP protein expression. To make it we exchanged the 5’UTR sequence of xylA-proD5'UTR in a biobrick [https://parts.igem.org/Part:BBa_K2014009 <b>(BBa_K1741009)</b>] to a synthetic, unstructured M5’UTR (picture B) derived from the Mel2 promoter [https://parts.igem.org/Part:BBa_K1741004 <b>(BBa_K1741004)</b>], which we provided to iGEM Registry in 2015.
+If you are interested in the pathway of our modifications please click here-> [https://static.igem.org/mediawiki/parts/c/c4/BBa_K2014004.pdf MODIFICATIONS OF XYLOSE INDUCED PROMOTERS]
+Biobricks used in description: <br><br>
+xylF-xylA - briefly called XylWT  [https://parts.igem.org/Part:BBa_K1741007 <b>(BBa_K1741007)</b>]<br>
+xylF-xylA-proD5'UTR - briefly called XylA1 [https://parts.igem.org/Part:BBa_K1741008 <b>(BBa_K1741008)</b>]<br>
+xylA-proD5'UTR - briefly called XylS  [https://parts.igem.org/Part:BBa_K1741009 <b>(BBa_K1741009)</b>]<br>
+xylS-M5'UTR - briefly called XylS-UTR [https://parts.igem.org/Part:BBa_K2014004 <b>(BBa_K2014004)</b>]<br>
+{|align="center"
+ |-valign="top"
+ | colspan = 2 | [[Image:BBa K2014004-1.png|thumb|500px|center|<font size="2"><b>Figure 1 Synthetic evolution of <i>E.  coli</i> xylose induced promoters in our lab up to xylS-UTR.</b> The promoter pxylS-M5'UTR (XylS-UTR) contains only xylA part of <i>E. coli</i> double sided xylose operon promoter, since xylF part appeared to be very weak. The synthetic, unstructured M5’UTR containing a strong, well-positioned RBS slightly enhances the responsiveness of xylA promoter to xylose. Despite that the promoters are stronger, they are still tight (Figure 2.).  </font>]]
+|}
+{|align="center"
+ |-valign="top"
+ | colspan = 2 | [[Image:BBa K2014004-2.png|thumb|500px|center|<font size="2"><b>Figure 2</b> Comparison of xylose promoters tightness during overnight time course cultures of <i>E. coli</i> DH5α in 1xLB medium without xylose. Bacteria were transformed with constructs: XylWT (xylF-xylA->sfGFP; [https://parts.igem.org/Part:BBa_K1741007 <b>BBa_K1741007</b>]), XylS (xylA-proD5'UTR; [https://parts.igem.org/Part:BBa_K1741009 <b>BBa_K1741009</b>]), XylS-UTR (xylS-M5'UTR; [https://parts.igem.org/Part:BBa_K2014004 <b>BBa_K2014004</b>]) and XylS-lysozyme (the construct containing non fluorescent protein- lysozyme, under XylS protein), which was used as our background. </font>]]
+|}
+{|align="center"
+ |-valign="top"
+ | colspan = 2 | [[Image:BBa K2014004-3.png|thumb|400px|center|<font size="2"><b>Figure 3 <br>A)</b> The most likely secondary structure that can be folded from the 5’UTR of XylWT promoter. <b>B)</b> The most likely secondary structures of M5’UTR from Mel2 and XylS promoters [https://parts.igem.org/Part:BBa_K1741004 <b>(BBa_K1741004)</b>]. The sequence of M5’UTR is designed to minimize the likelihood of secondary structure formation. Structures shown above, were generated by RNAFold (http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi)  under the same parameters. RBS sequences are underlined with a green line. </font>]]
+|}
+{|align="center"
+ |-valign="top"
+ | colspan = 2 | [[Image:BBa K2014004-5.png|thumb|700px|center|<font size="2"><b>Figure 4 <br>A)</b> Xylose inducibility of four xyl promoters/UTRs during 6h time course cultures of <i>E. coli</i> DH5α in LB medium containing 0.4% xylose. The efficiency of the improved promoter pxylS-M5'UTR ([https://parts.igem.org/Part:BBa_K1741004 <b>BBa_K2014004</b>]) is slightly higher, than all other versions of xylose responsive promoters we tested, and its induction seems to be faster (see 2h time-point). <b>B)</b> The growth rate of all constructs compared: Xyl-WT [https://parts.igem.org/Part:BBa_K1741007 <b>(BBa_K1741007)</b>], XylA1 [https://parts.igem.org/Part:BBa_K1741008 <b>(BBa_K1741008)</b>], XylS [https://parts.igem.org/Part:BBa_K1741009 <b>(BBa_K1741009)</b>] or XylS-UTR [https://parts.igem.org/Part:BBa_K2014004 <b>(BBa_K2014004)</b>] is very similar. </font>]]
+|}