Latest revision as of 21:24, 28 October 2013

Shuttle vectors for Lactobacillus and E. coli

Building bridges between E. coli and Lactobacillus

A shuttle vector is a plasmid that can be transferred between two different species and is able to replicate in both. Such a vector is key for bioengineering in Lactobacillus. The low transformation frequencies of ligations and longer generation times slows down the work pace tremendously if you have to assembly and test everything in Lactobacillus. A solution to this problem is if you instead build everything in E. coli and then transfer the finished construct to Lactobacillus. We have created two shuttle vectors that we have sent to the registry.

Shuttle vectors

BBa_K1033206, with chloramphenicol resistance.
BBa_K1033207, with erythromycin resistance.

Construction of shuttle vectors

To create the shuttle vector we replaced the replicon in a biobrick compatible plasmid BBa_K864003 with a replicon, pSH71, from a plasmid known to work in both E. coli and Lactobacillus. pSH71 originates from a plasmid, pJP059, from Lactococcus lactis but it is well known to replicate in both the Lactobacillus genus and in E. coli through rolling circle replication.^[1]

Many species in the Lactobacillus genus have inherent antibiotic resistances.^[2], because of that we have been limited in our selection of antibiotic resistance. Most commonly used are chloramphenicol and erythromycin and we made versions of both for our shuttle vector. BBa_K1033207 contains an erythromycin cassette from pLUL631 and BBa_K1033206 contains the resistance cassette from pSB1C3 but has had its promoter replaced with our cp29 promoter.^[3]

Results

We have managed to transform both shuttle vectors to E. coli and verified them by sequencing. We have also managed to subclone with them and replaced the red insert with chromoproteins expressed by our CP promoters that works both in E. coli and Lactobacillus, clearly yielding blue colonies.

To the left, BBa_K1033207 transformed to Lactobacillus reuteri 100-23, to the right, negative control without plasmid

BBa_K1033207 and RFP, BBa_J04450
insert in E. coli

BBa_K1033206 with a subcloned chromoprotein BBa_K1033282 in E. coli

We have also gained positive results when transforming BBa_K1033207 to Lactobacillus reuteri and Lactobacillus plantarum.

Reference

[1] Construction of compatible wide-host-range shuttle v... [Plasmid. 2001] - PubMed - NCBI." National Center for Biotechnology Information. N.p., n.d. Web. 1 Oct. 2013.

[2] "High efficiency recombineering in lactic acid bacteria. National Center for Biotechnology Information. N.p., n.d. Web. 1 Oct. 2013.

[3] Transformation of Lactobacillus reuteri with electroporation: Studies on the erythromycin resistance plasmid pLUL631 , Siv Ahrné, Göran Molin, Lars Axelsson, Januari 1992

Retrieved from "http://2013.igem.org/Team:Uppsala/vectors"

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                                                  <li><a href="https://2013.igem.org/Team:Uppsala/metabolic-engineering">Metabolic engineering</a>
                                                      <ul>
-                                                                 <li><a href="https://2013.igem.org/Team:Uppsala/p-coumaric-acid">P-coumaric acid</a></li>
+                                                                 <li><a href="https://2013.igem.org/Team:Uppsala/p-coumaric-acid">p-Coumaric acid</a></li>
                                                                  <li><a href="https://2013.igem.org/Team:Uppsala/resveratrol">Resveratrol</a></li>
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 					<ul>
-						<li><a href="https://2013.igem.org/Team:Uppsala/P-Coumaric-acid-pathway">P-Coumaric acid</a></li>
+						<li><a href="https://2013.igem.org/Team:Uppsala/P-Coumaric-acid-pathway">Kinetic model</a></li>
 						<li><a href="https://2013.igem.org/Team:Uppsala/modeling-tutorial">Modeling tutorial </a></li>
+<li><a href="https://2013.igem.org/Team:Uppsala/toxicity-model">Toxicity model</a></li>
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+                                                <li><a href="https://2013.igem.org/Team:Uppsala/advisors">Advisors</a></li>
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                                                  <li><a href="https://2013.igem.org/Team:Uppsala/Outreach">High school & media </a></li>
+<li><a href="https://2013.igem.org/Team:Uppsala/bioart">BioArt</a></li>
+<li><a href="https://2013.igem.org/Team:Uppsala/LactonutritiousWorld">A LactoWorld</a></li>
+<li><a href="https://2013.igem.org/Team:Uppsala/killswitches">Killswitches</a></li>
+<li><a href="https://2013.igem.org/Team:Uppsala/realization">Patent</a></li>
 					</ul></li>
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                                          <li><a href="https://2013.igem.org/Team:Uppsala/safety-form">Safety form</a></li>
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 	<div id="main_content"> <!-- Put content here -->
-	<h1 class="main-title">Vectors</h1>
+	<h1 class="main-title">Shuttle vectors for Lactobacillus and E. coli</h1>
 <div id="p-com-text1">
-<h1>Resveratrol (3,5,4′-trihydroxystilbene)</h1>
+<h1>Building bridges between E. coli and Lactobacillus</h1>
-		<table id="resveratrol-text1">
-		<td id="p-text">
-		<p>In our search for substances to put in our yoghurt we looked around in articles for molecules with good health effects. From the start we found resveratrol to stand out as a well-studied substance with some huge potential benefits. We mainly consume it via red wine and it is perhaps the main reason for why wine is considered good for your health in reasonable amounts.<br><br>
+		<p>A shuttle vector is a plasmid that can be transferred between two different species and is able to replicate in both. Such a vector is key for bioengineering in Lactobacillus. The low transformation frequencies of ligations and longer generation times slows down the work pace tremendously if you have to assembly and test everything in Lactobacillus. A solution to this problem is if you instead build everything in E. coli and then transfer the finished construct to Lactobacillus. We have created two shuttle vectors that we have sent to the registry.</p><br>
-Resveratrol belongs to a group of molecules known as Phyloalexin which is used by many plants to battle infections of all sorts.
-Since the early 90’s there has been many studies done on resveratrol showing it has a wide range of beneficial properties ranging from skin cancer reduction to anti-inflamatory effects and antioxidant properties.<sup><a href="#ref_point">[1]</a></sup><br><br>
-<sup><a href="#ref_point">[1]</a></sup>
+<h3>Shuttle vectors</h3>
+<a href="http://parts.igem.org/Part:BBa_K1033206">BBa_K1033206, with chloramphenicol resistance.</a><br>
+<a href="http://parts.igem.org/Part:BBa_K1033207">BBa_K1033207, with erythromycin resistance.</a>
+<br><br>
-Perhaps the most interesting property is that of life extension. With 2.1 billion people being overweight worldwide obesity related diseases are one of humanities greatest health problems today.
-Researchers now believe resveratrol is an caloric restricting substance reducing the effect of high caloric food and therefore increasing life expectancy.<sup><a href="#ref_point">[2]</a></sup>
-</p>
-		</td>
-		<td><img id="resveratrol" src="https://static.igem.org/mediawiki/2013/b/b6/Uppsala_Resveratrol.png"></td>
-		</table>
 </div>
 <div class="text">
-   <a id="b1"><h1>Methods</h1></a>
+   <a id="b1"></a><h1>Construction of shuttle vectors</h1>
    <p>
-   	Three enzymes is needed to produce resveratrol. Here we have characterized two of them, 4 coumarate ligase and stilbene synthase. For the first enzyme in the pathway, see the page for  <a href="https://2013.igem.org/Team:Uppsala/p-coumaric-acid">tyrosine ammonia lyase.</a><br><br>
+   	To create the shuttle vector we replaced the replicon in a biobrick compatible plasmid BBa_K864003 with a replicon, pSH71, from a plasmid known to work in both E. coli and Lactobacillus. pSH71 originates from a plasmid, pJP059, from Lactococcus lactis but it is well known to replicate in both the Lactobacillus genus and in E. coli through rolling circle replication.<sup><a href="#ref_point">[1]</a></sup><br><br>
--coumarate ligase (4Cl) and stilbene synthase (STS) genes from arabidophsis thaliana and vitis vinifiera was obtained from J.Conrado et al.<sup><a href="#ref_point">[3]</a></sup>. We biobricked 4Cl and STS with the ribosome binding site B0034 and overhangs in a single pcr. We also made a version with 6-HIS-tag for enzyme expression analysis. After biobricking the two enzymes, we coupled them together to create an operon consisting of 4Cl and STS. <br><br>
+Many species in the Lactobacillus genus have inherent antibiotic resistances.<sup><a href="#ref_point2">[2]</a></sup>, because of that we have been limited in our selection of antibiotic resistance. Most commonly used are chloramphenicol and erythromycin and we made versions of both for our shuttle vector. BBa_K1033207 contains an erythromycin cassette from pLUL631 and BBa_K1033206 contains the resistance cassette from pSB1C3 but has had its promoter replaced with our cp29 promoter.<sup><a href="#ref_point3">[3]</a></sup><br><br> </p>
-We have expressed 4Cl and STS in e-coli DH5alpha and E. Coli nissle, a probiotic E. coli obtained from Trieste iGEM 2012. 4Cl and STS will also be characterized in lactobacillus, by transforming the construct with our shuttle vector.<br><br>
-  </p>
    </div>
-   <img class="hej" src="https://static.igem.org/mediawiki/2013/d/d1/Uppsala_4CL-STS.png">
+   <a href="https://static.igem.org/mediawiki/2013/9/92/Shuttle-vector_pSBLbC.png" data-lightbox="roadtrip"><img class="vector-plasmid" src="https://static.igem.org/mediawiki/2013/9/92/Shuttle-vector_pSBLbC.png"></a>
 <h1>Results</h1>
-<h3>Summary</h3>
-Although we managed to clone out and sequence verify the genes for resveratrol production, we have had some problems in the characterization. The results are unclear, and we did not have time for further investigations.For detailed information about the characterisation methods, see the <a href="https://2013.igem.org/Team:Uppsala/protocols">protocol</a> section <br><br>
-<h3>Biobrick</h3>
-We succeded in the cloning and sequencing of our two biobricks, 4-Coumarate ligase from
-arabidophsis thaliana and Stilbene synthase from vitis vinifiera with the RBS B0034 that should work in various organisms, lactobacillus and e-coli. Sequencing was done at GATC biotech and Uppsala Genome center using sanger sequencing. <br><br>
-We also succeded in the making of our operon containing 4Cl and STS together.<br><br>
+<p>We have managed to transform both shuttle vectors to E. coli and verified them by sequencing. We have also managed to subclone with them and replaced the red insert with chromoproteins expressed by our CP promoters that works both in E. coli and Lactobacillus, clearly yielding blue colonies.</p>
-<li> <a href="http://parts.igem.org/Part:BBa_K1033000">BBa_K1033000</a> - tyrosine ammonia lyase (TAL) with RBS </li>
+<div id="pic_type2">
-<li> <a href="http://parts.igem.org/Part:BBa_K1033001">BBa_K1033001</a> - 4 coumarate ligase (4CL) with RBS </li>
+<a href="https://static.igem.org/mediawiki/2013/e/e1/IMG_6644.JPG" data-lightbox="roadtrip" title="BBa_K1033207 transformed to Lactobacillus reuteri 100-23"><img class="pic_type2" src="https://static.igem.org/mediawiki/2013/e/e1/IMG_6644.JPG"></a>
-<li> <a href="http://parts.igem.org/Part:BBa_K1033002">BBa_K1033002</a> - stilbene synthase (STS) with RBS </li>
-<li> <a href="http://parts.igem.org/Part:BBa_K1033003">BBa_K1033003</a> - B0034-4Cl-B0034-STS </li>
-<br>
+<i>To the left, BBa_K1033207 transformed to Lactobacillus reuteri 100-23, to the right, negative control without plasmid</i>
+</div>
-<h3>Western blot</h3>
+<div id="pic_type3">
-We also succeeded in expressing the enzyme stilbene synthase in E. coli. Although our expression of the protein was very weak, and due to time constraints we were not able to optimize our experiment.  <br><br>
+<a href="https://static.igem.org/mediawiki/2013/2/28/Uppsala2013_chromo.JPG" data-lightbox="roadtrip" title="BBa_K1033207 and RFP, BBa_J04450 insert in E. coli"><img class="pic_type3" src="https://static.igem.org/mediawiki/2013/2/28/Uppsala2013_chromo.JPG"></a>
-To enable the detection of this protein by anti-his antibodies, 6-histidine tags was incorporated in the sequence. This way we could detect our enzyme with anti-his antibodies. <br><br>
+<i class="middle-text">BBa_K1033207 and RFP, BBa_J04450
+<br>
+<i class="middle-text">insert in E. coli</i>
+</div>
-We expressed our protein with a promotor working in both lactobacillus and e-coli. This way, we can
+<div id="pic_type4">
-easily transfer stilbene synthase to lactobacillus later on. <br><br>
+<a href="https://static.igem.org/mediawiki/2013/b/ba/Uppsala2013_Shuttle_Vector_pSBLBC_cp29_amilCP.JPG" data-lightbox="roadtrip" title="BBa_K1033206 with a subcloned chromoprotein BBa_K1033282 in E. coli"><img class="shuttle_vec" src="https://static.igem.org/mediawiki/2013/1/18/Uppsala2013_Shuttle_Vector_pSBLBC_cp29_amilCP1.png"></a>
-The size of our protein was calculated using ProtParam <sup><a href="#ref_point">[5]</a></sup>, 43 kDA. <br><br>
+<i>BBa_K1033206 with a subcloned chromoprotein BBa_K1033282 in E. coli</i>
+</div>
-<img id="sts_westp_uppsala" src="https://static.igem.org/mediawiki/2013/3/39/Resv_westblot_uppsala.png"><br>
-<i><b>Figure 1:</b>Number 2 shows a very weak band of our protein at around 43 kDA.<br>
-Positive control -> 1, Stilbene synthase -> 2</i><br><br><br>
-<h3>High pressure liquid chromatography</h3>
-We tested our biobrick 4Cl-STS on HPLC, by adding p-coumaric acid as a precursor. The result we saw was quite unclear. We saw that the e-coli produced something out of the ordinary, but the absorbation was low and the peaks did not exactly match the standard. The peak at around ~33 min could correspond to our standard, but it is unclear. We theorize it is something in the actual hplc measurement that fails, or that something happens to our resveratrol metabolite in our e-coli. This result could correspond to the poor results in our blot.  We hope that iGEM teams can continue to work on these biobricks in the future.   <br><br>
-<b>4Cl-STS translational fusion expressed in e-coli</b>
+<div id="clear"></div>
- <img class="results_pic_pc" id="Resveratrol_fig_1" src="https://static.igem.org/mediawiki/2013/d/dc/Resva_uppsala_tab1.png"><br>
-<i><b>Figure 1:</b> E. coli supposed to produce resveratrol. As we can see, we got very low absorbance peaks at ~30 min, ~33 min and ~36 min. </i><br><br><br>
-<b>Resveratrol standard</b>
+<p>We have also gained positive results when transforming BBa_K1033207 to Lactobacillus reuteri and Lactobacillus plantarum. <p>
- <img class="results_pic_pc" id="Resveratrol_fig_2" src="https://static.igem.org/mediawiki/2013/3/33/Resva_uppsala_tab2.png"><br>
-<i><b>Figure 2:</b> Resveratrol standard, peaks around ~33, ~34 min. </i><br><br><br>
-<b>Resveratrol standard scaled</b>
- <img class="results_pic_pc" id="Resveratrol_fig_3" src="https://static.igem.org/mediawiki/2013/8/82/Resva_uppsala_tab3.png"><br>
-<i><b>Figure 3:</b>  Resveratrol standard that is scaled down to correspond to the absorbations of our e. coli supposed to produce the corresponding metabolite. The peaks are at around ~33 and ~34. </i>
-<br><br><br>
-<a name="ref_point"></a>
-<b>Lysed bacterial culture without plasmid of assembly</b><br>
-<img class="results_pic_pc" src="https://static.igem.org/mediawiki/2013/4/4f/Uppsala_char_coumaric-acid_blank.png"><br>
-<i><b>Figure 3:</b> E. coli culture injected to the hplc without our biobrick tyrosine ammonia lyase. Here we can see that there is originally no peaks around 30-35 minutes.</i> <br><br><br>
-  <h1></h1>
-		<div id="reference">
+<div id="reference">
+<h1>Reference</h1>
 			<p class="reference">
-<a>[1]</a> Sinclair, D. A & Baur, Y, A. Therapeutic potential of resveratrol:
+<a id="ref_point">[1]</a> <a href="http://www.ncbi.nlm.nih.gov/pubmed/11591136"> Construction of compatible wide-host-range shuttle v... </a>  [Plasmid. 2001] - PubMed - NCBI." National Center for Biotechnology Information. N.p., n.d. Web. 1 Oct. 2013.
-the in vivo evidence. Nature 506 | JUNE 2006 | VOLUME 5<br><br>
-<a>[2]</a> Baur, Y, A. et al. Resveratrol improves health and survival
+<br><br>
-of mice on a high-calorie diet. Nature Vol 444| 16 November 2006<br><br>
-<a>[3]</a> Robert J. Conrado et al, DNA guided assembly of biosynthetic pathways promotes improved catalytic effiency. Nucleic Acids Research , 2012, Vol 40 NO 4, 1879-1889 <br><br>
+<a id="ref_point2">[2]</a> <a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3378904"> "High efficiency recombineering in lactic acid bacteria.  </a> National Center for Biotechnology Information. N.p., n.d. Web. 1 Oct. 2013.
+<br><br>
+<a id="ref_point3">[3]</a> <a href="http://www.researchgate.net/publication/226939823_Transformation_ofLactobacillus_reuteri_with_electroporation_Studies_on_the_erythromycin_resistance_plasmid_pLUL631?ev=pub_cit%22,%22http://www.researchgate.net/publication/226939823_Transformation_ofLactobacillus_reuteri_with_electroporation_Studies_on_the_erythromycin_resistance_plasmid_pLUL631?ev=pub_cit%22)
+"> Transformation of Lactobacillus reuteri with electroporation: Studies on the erythromycin resistance plasmid pLUL631 </a> , Siv Ahrné, Göran Molin, Lars Axelsson, Januari 1992
-			<a>[4]</a> Sinclair, D. A & Baur, Y, A. Therapeutic potential of resveratrol:
-the in vivo evidence. Nature 506 | JUNE 2006 | VOLUME 5
-<br><br>
-			<a>[5]</a> Baur, Y, A. et al. Resveratrol improves health and survival
-of mice on a high-calorie diet. Nature Vol 444| 16 November 2006
 <br><br>
-<a>[6]</a> Here's a link to ProtParam: <a href="http://web.expasy.org/protparam/">http://web.expasy.org/protparam/</a><br><br>
 </p>
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