Team:UC Chile/Cyanolux/Results

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<h1>Construction of plasmids</h1>
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<h1>Plasmid Construction</h1>
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<h2>pSB1C3_IntK</h2>
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Following our experimental strategy we built two plasmid backbones on which to insert the Lux constructs.
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Our first attempts to build the construct ([[http://partsregistry.org/Part:BBa_K743004| BBa_K743004]]) through Gibson Assemly were unsuccessful due to problems involving high compexity of the reaction, however we were able to Biobrick the recombination sites ([[http://partsregistry.org/Part:BBa_K743000 | Part:BBa_K743000]] and [http://partsregistry.org/Part:BBa_K743001 | Part:BBa_K743001]). Afterwards we decided to build a simple backbone first before continuing with more complex assemblies.
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<h2>Neutral recombination plasmid</h2>
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Through standard assembly we managed to build [http://partsregistry.org/Part:BBa_K743006| K743006] which led us to continue assembling our constructs through simpler Gibson Assemblies.  
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[[File: C4_Digestion.jpg|400px]]
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1st lane is 1Kb Ladder. 4th lane is C4; Band at 2300: Digestion intermediate, band at 2000: backbone, band at 1700: RS1-KanR-RS2partial, band at 500: RS2partial.
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[[File: C4_Digestion.jpg| 300px| right]]
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This plasmid backbone was constructed by standard assembly techniques. Recombination sites ([http://partsregistry.org/Part:BBa_K743000 BBa_K743000] and [http://partsregistry.org/Part:BBa_K743001  Part:BBa_K743001]) were Biobricked by Gibson assembly and were consenquently ligated to neighboring parts and  KanRB0015 in parallel assembly. Afterwards, we ligated both composites through a standard assembly reaction. Final construct was validated by digestion (see gel image at right) and corroborated through sequencing.
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<h3>sfGFP with LVA tag for describing circadian behaviour</h3>
 
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To describe the circadian behaviour of the promoter we built a fast-degrading reporter consisting of sfGPF I746916 with a LVA degradation tag in the C-terminal end of the protein. This construct will serve as a real-time reporter of promoter activity, you may find more information about the half-life of proteins with the LVA tag  [http://partsregistry.org/wiki/index.php?title=Part:BBa_M0050 | here]. The construct has been verified by digestion and corroborated by sequencing.
 
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[[File: sfGFP.tag_digestion.jpg| 300px| right]]
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After obtaining [http://partsregistry.org/Part:BBa_K743006 BBa_K743006], we proceeded assembling our constructs through Gibson assemblies.
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<h3>Luciferase(s)</h3>
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In short time we were able to build our final constructs for the luciferase using 2 different versions of it available in the registry ([http://partsregistry.org/Part:BBa_K743014 | From Photorhabdus luminiscent, BBa_K743014] and [http://partsregistry.org/Part:BBa_K743015 | from Vibrio fisherii, BBa_K743015]) under an endogenous  Synechocystis's promoter (transaldolase Reference???). All constructs and parts have been verified by digestion and corroborated by sequencing.
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[[File: LuxABxl_digestion.jpg| 300px| right]]
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<i>If you are wondering why the plasmid was constructed by standard assembly and not by gibson assembly from scratch, please refer to </i> [[Team:UC_Chile/Results/Gibson | Gibson assembly for small parts ]]
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[[File: LuxABvf_digestion.jpg| 300px| right]]
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<h2>pSB1A3_IntC (From 2010 Utah's iGEM team)</h2>
 
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We decided to use this plasmid to place the LuxCDEG part of the Lux operon in this plasmid, however after various attempts to transform Synechocystis without success we reconsidered (see below SOMEWHERE!!! [[]]) due to problems regarding the design of this plasmid backbone. After analyzing the sequence of the vector (CODE HERE) we blasted the sequences of the recombination sites and we found out that position of the sequences of each recombination site are swapped, leaving the backbone part of the plasmid to be integrated into the genome. Furthermore, the recombination sites are separated by aproximately 2.2 kilobases, knocking 1 gene.
 
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[[File: Construct Recombination sites position.jpg| 500px| center]]
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<h3>Insertion of LuxAB genes</h3>
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Starting from [http://partsregistry.org/Part:BBa_K743006| K743006] as a plasmid backbone, we were able to build our LuxAB constructs for the bacterial luciferase using two different versions available at the registry (from [http://partsregistry.org/Part:BBa_K743014 Photorhabdus luminiscent, BBa_K743014] and [http://partsregistry.org/Part:BBa_K743015 Vibrio fisherii, BBa_K743015]) under an endogenous Synechocystis's promoter (transaldolase Reference???). Resulting constructs were verified by digestion (see gel images below) and corroborated by sequencing.
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You can see their original construct design [https://2010.igem.org/Design_usu.html#Overview_of_Integration| here].
 
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[[File: Position in genomic DNA.jpg| 300px| right]]
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[[File: LuxABxl_digestion.jpg| 400px| right]]
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Lane 1 = Ladder 1Kb, Lane 6 = K743014. Band at 4000: RS1-Pta-LuxABvf-Kan-RS2partial, band at 2000: pSB1C3, band at 500: RS2partial.
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[[File: LuxABvf_digestion.jpg| 366px| left]]
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Lane 1 = Ladder 1Kb, Lane 3 = K743014. Band at 4000: RS1-Pta-LuxABxl-Kan-RS2partial, band at 2000: pSB1C3, band at 500: RS2partial.
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[File:UC_Chile-CN2Y_Possible_Recombination.jpg]]
 
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[[File:UC_Chile-CN2Y_Possible_Recombination_Results.jpg]]
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[[File:UC_Chile-INT_C_Febugging.jpg]]
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<h2>Suceptibility construct</h2>
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<h2>pSB1C3_IntS</h2>
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After we reconsidered about using pSB1A3_IntC we designed a new plasmid backbone to place the LuxCDEG (substrate regeneration part of the Lux operon) under the Synechocystis's promoters we choose through our modelling (LINK HERE). We have obtained the plasmid backbone including all parts through Gibson Assembly and the plasmid has validated through digestion and was corroborated through sequencing.
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[[File: Position in genomic DNA.jpg| 300px| right]]
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pSB1C3_IntS was constructed in two successive Gibson assemblies. The first assembly inserted the CopS region (the entire RS5 & RS6 recombination locus) to a chopped-down pSB1C3 lacking the whole VF2 to VR region. The second assembly inserted the Spectynomycin resistance selection marker and the whole VF to VR region of plasmid pSB4K5-J04450 right in between of the RS5 & RS6 CopS region. This construct yields red colonies if correctly assembled (see image at left). Red colonies were colony PCRed, verificated by digestion (see image down right) and corroborated by sequencing.
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<h3>Substrate production under Pcaa3</h3>
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We have assembled all parts of the substrate production pathway ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K325902| LuxCD] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K325903| LuxEG]) for the final construct under the Pcaa3 promoter (which has also been Biobricked with code [http://partsregistry.org/wiki/index.php?title=Part:BBa_K743002 K743002] and we are now verifying for positive assembles.
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[[File: K743010.jpg| 400px| left]]
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A couple of picked red colonies are shown in this plate.  
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<h3>Substrate production under PsigE</h3>  
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As this assembly was unsuccessful previously, we are re-amplifying the LuxCD part (to obtain higher concentrations of the insert) and we will retry the assembly after the wiki freeze.
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[[File: C10 digestion.jpg| 400px| right]]
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Digestion profile for [http://partsregistry.org/Part:BBa_K743018 K743018] & [http://partsregistry.org/Part:BBa_K743010 K743010 ]
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</div>
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<h1>Synechocystis</h1>
 
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<h2>Standardizing growth conditions</h2>
 
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While we managed to acquire an inoculum of Synechocystis PCC 6803, no conditions were set for working with Synechocystis PCC 6803 in our faculty. We proceeded to standardize our own growth conditions by building a custom made cyanobacterial incubator based on growth conditions described in the literature (References?)
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<h3>Insertion of Lux substrate biosynthesis genes under twilight promoters</h3>
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We have amplified all the parts of the substrate production pathway ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K325902 LuxCD] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K325903 LuxEG]) for the final construct under the Pcaa3 promoter (which has also been Biobricked with code [[http://partsregistry.org/wiki/index.php?title=Part:BBa_K743002 K743002]]. Also, we are amplifying one remaining part (LuxCD with 5' homology to PsigE) to do Gibson assembly of the LuxCDEG construct under PsigE. Gibson reaction will be done along with the substrate production construct under Pcaa3 in pSB1C3_IntS for both constructs.
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<h1>Synechocystis PCC 6803</h1>
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<h2>Transforming Synechocystis PCC 6803</h2>
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Once constructs were corroborated by sequencing, we proceeded to [[Team:UC_Chile/Protocols#Transformation_of_Synechocystis_PCC._6803 | transform synechocystis]]. Prior to transforming, we verified that our Synechocystis cultures were in exponential growth by comparison to our [[Team:UC_Chile/Results/Growthcurve | growth curve characterization]].
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Here we show our growth curve for Synechocystis PCC 6803 using the conditions set in our [[Team:UC_Chile2/DIY | DIY]]) shaking incubator (40uE/s/m²), 90 RPM, 30°C.
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Transformation in Synechocystis takes about two weeks to reveal transformant colonies, which in turn have to be restreaked again before PCR verification. This makes working in Synechocystis pressing as mistakes [[Team:UC_Chile/Results/Int_C | can take long to unravel]].
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[[File:ColoniasChilenas.jpg | 600px | center]]
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This image shows various time points during a Synechocystis PCC. 6803 transformations. Be advised that it is only to be used as a reference of what to expect during the first week of transformation. 
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<h3>LuxABxl under transaldolase promoter transformation</h3>
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[[File:Pta_LuxABxl.JPG| 600px| center]]
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Transformation of Synechocystis PCC 6803 with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K743014 K743014] on the 14th day of transformation. The left plate is the control transformation plate (no DNA) on Kanamycin 25ug/mL and on the right, successfully transformed Synechocystis colonies growing throughout the Millipore membrane on the same antibiotic.
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<h3>LuxABvf under transaldolase promoter transformation</h3>
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[[File:Pta_LuxABvf.JPG| 600px| center]]
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Transformation of Synechocystis PCC 6803 with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K743015 K743015] on the 14th day of transformation. The left plate is the control transformation plate (no DNA) on Kanamycin 25ug/mL and on the right, successfully transformed Synechocystis colonies growing throughout the Millipore membrane on the same antibiotic.
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<h2>Corroboration of integration</h2>
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To corroborate if the transformant colonies had integrated the actual insert, we did colony PCR for multiple parts contained in the insert.
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<h3>Integration of LuxABxl under transaldolase promoter</h3>
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We did a massive colony PCR to corroborate the presence of the parts which composed the original K743014 integration plasmid, with corresponding negative controls to avoid confusing a genomic amplicon with an insert amplicon.
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Regretably we had many negative controls which had amplicons and also lots of amplicons of the insert which we could not obtain... We are not certain about the repercussions of this result so we will try the experiment after the wiki freeze.
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Methods for the growth curve characterization of Synechocystis PCC 6803 over [[Team:UC_Chile2/Protocols#SyneGrowth | here]].
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<h3>Integration of LuxABvf under transaldolase promoter</h3>
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[[File:LuxABvf colony PCR.JPG| 400px| center]]
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This PCR was done using DNA from transformant LuxABvf colonies to amplify parts contained in the insert in lanes 2,4,6,8 and DNA from wild-type Synechocystis PCC 6803, used as a negative control in lanes 3,5,7,9. Ladder 1Kb is placed in lane 1 and 10.
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[[File: Figure for the growth curve.jpg| 500px |center]]
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<p style="font-size:150%">We would like to make notice that to our knowledge we are the first iGEM team to do a succesful direct transformation of Synechocystis PCC. 6803 with naked DNA!</p>
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<h2>Transforming Synechocystis PCC 6803</h2>
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<h2>Testing constructions in Synechocystis</h2>
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To test our constructions, we managed to get access to a Luminometer which also has dispensing capabilities. To test the activity of the transformed Synechocystis with the luciferases we used decanal and dodecanal exogenously, using incremental concentrations of substrate.
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[[File:decanalxp.jpg| 400px | left]]
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According to these results we are inclined to think that our the transaldolase promoter that we are using is not driving the expression of the luciferases. Furthermore, we have received recent advice to use much larger (up to 1 Kb) of promoter upstream of the +1 transcription site of the transaldolase gene.
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After corroborating our constructs we proceded with the [[Team:UC_Chile/Protocols#Transformation_of_Synechocystis_PCC._6803 | Synechocystis transformation]] for each of the constructs as soon as we obtained sufficient concentration (10ug) of the construct.
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Transformation in Synechocystis takes about 2 weeks to reveal transformant colonies, which in turn have to restreaked again before PCR verification. This makes working in Synechocystis pressing as mistakes can take long to unravel.
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<h2>sfGFP with degradation tag to characterize transaldolase promoter</h2>
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To describe the circadian behaviour of the transaldolase promoter without having the dificulties of equipment (luminometer and substrate), we built a fast-degrading reporter consisting of sfGPF(I746916) with a LVA degradation tag in the C-terminal end of the protein,  [http://partsregistry.org/Part:BBa_K743019 BBa_K743019]. This construct will serve as a real-time reporter of promoter activity. As it is a reporter plasmid with fast-degradation tags, verificating if sfGFP is being expressed circadianly by Synechocystis should be effortless.
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<h3>BBa_K743009</h3>
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You may find more information about the half-life of proteins with the LVA tag  [http://partsregistry.org/wiki/index.php?title=Part:BBa_M0050 | here]. The construct has been verified by digestion and corroborated by sequencing. Check construct digestion [[Team:UC_Chile/Cyanolux/Results#C10 | here]]
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[[File:C1RCHILE.JPG|500px| right]]
 
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This picture shows a transformation after 11 days. On the left there is the control transformation plate (no DNA) on antibiotic and in the right a positive transformation of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K743009 K743009] with its corresponding Synechocystis colonies growing througout the Millipore membrane on the same antibiotic.
 
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<h3>[[File:C1.2R.JPG|500px| right]]
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<h3>High-sensitive camera</h3>
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<h2>Reestreaking</h2>
 
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During the Latin America Jamboree, we had a chat with a couple of judges and a student from the Universidad de los Andes, David Olarte, on how to induce Synechocystis with n-decanal.
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<h2>Confirming DNA</h2>
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David sent us some work he had done on luminescence assays in cyanobacteria. Following his methods we finally were able to see light emittion, confirming prescence of catalytically active luciferase.
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<html><center><img src="https://static.igem.org/mediawiki/2012/1/1e/Results.post.colombia.camera.jpg" align="left" width="700"></center></html>
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<h2>Conclusions</h2>
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<h1>Experimental Highlights</h1>
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- Constructed two functional integration plasmids for <i>Synechocystis</i> PCC. 6803
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- Transformed <i>Synechocystis</i> PCC. 6803 with an optimized transformation protocol, available [https://2012.igem.org/Team:UC_Chile/Protocols#Transformation_of_Synechocystis_PCC._6803 here]
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- Verified integration of constructs
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- Confirmed expression of genes and activity of luciferase through a bioluminescence assay with exogenous substrate.
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<h1>Revealing phenotypes</h1>
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<h1>Conclusions</h1>
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<h2>Transformation in Synechocystis PCC. 6803</h2>
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We have successfully transformed Synechocystis PCC. 6803. We have been able to standarize growth conditions and transformation protocols. We believe that we are the first iGEM team to transform  Synechocystis PCC. 6803 directly with naked DNA.
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<h2>Microscopy</h2>
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<h2>Verification of integrations</h2>
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We have been able to verify the integration of the construct into Synechocystis's genome.
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<h2>Promoters</h2>
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We have been able to demonstrate that the transaldolase is driving the expression of the constructs but we have no certainty as if it is behaving circadianly. With our sfGFP construct with degradation tag we will be able to determinate if that is so.
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<h2>Luminometer readings</h2>
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<h2>Luciferases</h2>
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With our new results with the High-sensitivity CCD camera we have been able to observe bioluminescence from our transformed Synechocystis. The luciferase is catalytically active, however we need to see if we can enhance light output by changing the length of the promoter.
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<h2> Susceptibility construct</h2>
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We have been unable to obtain a correct assembly of the LuxCDEG (substrate production) into our suceptibility construct. Due to repetitive failures, we will try a new strategy of cloning, by ordering new primers with longer overlapping ends.
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<h2>Conclusions</h2>
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Latest revision as of 10:05, 26 October 2012

Project: Luxilla - Pontificia Universidad Católica de Chile, iGEM 2012