diff --git a/wiki/pages/attributions.html b/wiki/pages/attributions.html
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+++ b/wiki/pages/attributions.html
@@ -44,7 +44,7 @@
<h1>Project support and advice</h1>
<p>Members of the Synbio club: participated in outreach activities with the iGEM team.</p>
- <p>Prof. Hernane Barud team (Uniara): provided Komagataeibacter strains and training on cultivation and Bacterial Cellulose production, in addition to valuable insights on how we could develop our project.</p>
+ <p>Prof. Hernane Barud team (Uniara): provided <i>Komagataeibacter</i> strains and training on cultivation and Bacterial Cellulose production, in addition to valuable insights on how we could develop our project.</p>
<p>Prof. Hernandes Carvalho´s team: cultivated fibroblast on Bacterial cellulose.</p>
<p>Prof. Carmen Versissima´s team: cultivated mellanoma cells on Bacterial Cellulose.</p>
<p>Prof. Marcelo Brocchi´s team: granted us permission to use his electroporator.</p>
diff --git a/wiki/pages/contribution.html b/wiki/pages/contribution.html
index 99c1e99b93842829183dd4b6475d6632019c57e7..98fcafa92db90870cdb64c8a5595f3fb7ac26248 100644
--- a/wiki/pages/contribution.html
+++ b/wiki/pages/contribution.html
@@ -72,12 +72,12 @@
<ul>
<li>IBGE. Produção de Laranja.</li>
<li>Yanwen Wu. Software Engineering and Knowledge Engineering: Theory and Practice. Advances in Intelligent and Soft Computing (2019).</li>
- <li>Cannazza, P. et al. Characterization of komagataeibacter isolate reveals new prospects in waste stream valorization for bacterial cellulose production. Microorganisms 9, (2021).</li>
- <li>Corrêa dos Santos, R. A. et al. Draft genome sequence of Komagataeibacter rhaeticus strain AF1, a high producer of cellulose, isolated from Kombucha Tea. Genome Announc. v.2 n4 (2014).</li>
- <li>Corrêa dos Santos, R. A. et al. Draft genome sequence of Komagataeibacter intermedius strain AF2, a producer of cellulose, isolated from Kombucha Tea. Genome Announc. 3, 1–2 (2015).</li>
+ <li>Cannazza, P. et al. Characterization of <i>Komagataeibacter</i> isolate reveals new prospects in waste stream valorization for bacterial cellulose production. Microorganisms 9, (2021).</li>
+ <li>Corrêa dos Santos, R. A. et al. Draft genome sequence of <i>Komagataeibacter rhaeticus</i> strain AF1, a high producer of cellulose, isolated from Kombucha Tea. Genome Announc. v.2 n4 (2014).</li>
+ <li>Corrêa dos Santos, R. A. et al. Draft genome sequence of <i>Komagataeibacter intermedius</i> strain AF2, a producer of cellulose, isolated from Kombucha Tea. Genome Announc. 3, 1–2 (2015).</li>
<li>Gomes, Fabio P., et al. Production of bacterial cellulose by Gluconacetobacter sacchari using dry olive mill residue. Biomass and Bioenergy 55 (2013): 205-211.</li>
- <li>Jacek, P., Silva, F. A. G. S. da, Dourado, F., Bielecki, S. & Gama, M. Optimization and characterization of bacterial nanocellulose produced by Komagataeibacter rhaeticus K3. Carbohydr. Polym. Technol. Appl. 2, (2021).</li>
- <li>Machado, R. T. A. et al. Komagataeibacter rhaeticus as an alternative bacteria for cellulose production. Carbohydr. Polym. 152, 841–849 (2016).</li>
+ <li>Jacek, P., Silva, F. A. G. S. da, Dourado, F., Bielecki, S. & Gama, M. Optimization and characterization of bacterial nanocellulose produced by <i>Komagataeibacter rhaeticus</i> K3. Carbohydr. Polym. Technol. Appl. 2, (2021).</li>
+ <li>Machado, R. T. A. et al. <i>Komagataeibacter rhaeticus</i> as an alternative bacteria for cellulose production. Carbohydr. Polym. 152, 841–849 (2016).</li>
<li>Pacheco, Guilherme et al. Development and characterization of bacterial cellulose produced by cashew tree residues as alternative carbon source. Industrial Crops And Products. Amsterdam: Elsevier Science Bv, v. 107, p. 13-19, 2017.</li>
<li>Siqueira, M.U., Contin, B., Fernandes, P.R.B. et al. Brazilian Agro-industrial Wastes as Potential Textile and Other Raw Materials: a Sustainable Approach. Mater Circ Econ 4, 9 (2022).</li>
<li>Urbina, L., Corcuera, M.Ã., Gabilondo, N. et al. A review of bacterial cellulose: sustainable production from agricultural waste and applications in various fields. Cellulose 28, 8229–8253 (2021).</li>
diff --git a/wiki/pages/description.html b/wiki/pages/description.html
index 8f0186cdc78b89f4d9e1f6c030e0ae462fe5b80b..6b2cb5a852faa605f3decaff7be6ab06393ec08e 100644
--- a/wiki/pages/description.html
+++ b/wiki/pages/description.html
@@ -22,7 +22,7 @@
<p>Following the principles of green chemistry, we aim to up-cycle biomass generated in the massive agroindustrial production in Brazil, by giving it a useful destination, in contrast to discarding in the environment. We disseminate our work in schools so that new generations are inspired by science and its various contributions. With support, we are sure that Cellulopolis will achieve significant improvements in the medical treatment of burns in people regardless of their socioeconomic status and will enable the advancement of medical research using BC as a scaffold.</p>
<hr>
<h1>Bacterial Cellulose</h1>
- <p>Cellulose is a natural polymer present in the structure of cell walls throughout the plant, formed by a complex chain of sugars, including glucose. It is a very abundant material (33% of all plant biomass on the planet) and is intrinsically linked to various elements of everyday life, such as paper, diapers, fabrics, pills and even food. However, the processing of wood into cellulose requires large amounts of water and energy, in addition to the use of polluting chemicals, such as nitrogen and sulfur, which are released into the air. It is also worth noting that the purification of cellulose from plants is expensive and laborious, leading to the need to replace the production of this polymer with a more sustainable alternative.9</p>
+ <p>Cellulose is a natural polymer present in the structure of cell walls throughout the plant, formed by a complex chain of sugars, including glucose. It is a very abundant material (33% of all plant biomass on the planet) and is intrinsically linked to various elements of everyday life, such as paper, diapers, fabrics, pills and even food. However, the processing of wood into cellulose requires large amounts of water and energy, in addition to the use of polluting chemicals, such as nitrogen and sulfur, which are released into the air. It is also worth noting that the purification of cellulose from plants is expensive and laborious, leading to the need to replace the production of this polymer with a more sustainable alternative.</p>
<p>A good alternative to plant cellulose is bacterial cellulose, which has the same chemical structure as the plant counterpart, but without “contaminants†such as hemicellulose, lignin, and pectin. This easily moldable material has a three-dimensional network structure of cellulose nanofibrils in the form of a ribbon, which is capable of efficiently absorbing and retaining water.</p>
<p>This BC is produced by bacteria in a natural and purer way than by plants, being a very versatile natural biomaterial that can have its production optimized through bacterial engineering techniques. Bacteria can convert about 50% of carbon into cellulose, varying according to growth conditions, and naturally produce it for various purposes, such as protection and preservation of nutrients. The properties of cellulose vary according to the bacterium that produces it and the substrates available in the cultivation medium.</p>
<p>There are several possible applications for bacterial cellulose, including in the medical field, where it can be widely used in the treatment of wounds and burns, as artificial skin during healing. However, this process has a high cost and pulp production is not very efficient, making it necessary to seek measures for the development of modified strains in order to increase pulp production, and also to lower the costs involved in this process. Bacterial cellulose can also have applications in food, cosmetics, and bioethanol areas, not forgetting its use as a scaffold for the growth of cells, tissues, and organs.</p>
@@ -31,7 +31,7 @@
<h1>Inspiration</h1>
<p>The idea for a waste management project arose from research and brainstorming within the team, which started its journey in 2021, with an intense interest in making a real impact on the paths of synthetic biology in Brazil and the desire to transform waste into high-value products. Our goal until May 2021 was to engineer bacteria to digest polystyrene and convert it into PHA or PHB, in a project named Styropolis (video).</p>
<p>We designed a strategy whereby that goal could technically be achieved, however, through diverse insights obtained by stakeholders we learned that, even though polystyrene is recyclable and could potentially be used as a carbon source for bioplastic production, its low density leads to high transportation costs per kg of raw material, creating a negative environmental impact that would detract from our “green†goals. This is discussed in greater detail in the Human Practices section.</p>
- <p>Inspired by the Imperial_College iGEM team (<i>K. rhaeticus</i> iGEM strain sequencing and toolkit development) and by collaborators from BioSmart Nanotechnology, we decided to develop a strain capable of producing cellulose on a large scale utilizing based on Komagataeibacter rhaeticus AF1, through synthetic biology. The Cellulopolis project was born!</p>
+ <p>Inspired by the Imperial_College iGEM team (<i>K. rhaeticus</i> iGEM strain sequencing and toolkit development) and by collaborators from BioSmart Nanotechnology, we decided to develop a strain capable of producing cellulose on a large scale utilizing based on <i>Komagataeibacter rhaeticus</i> AF1, through synthetic biology. The Cellulopolis project was born!</p>
<p>BioSmart Nanotechnology is a research and development company that, in collaboration with JBT Corporation, works on the Food Waste project, which consists of the reuse of agroindustry residues to produce BC (a technology proprietary of the consortium Uniara, BioSmart Nanotechnology, and HB Biotec).</p>
<hr>
diff --git a/wiki/pages/design.html b/wiki/pages/design.html
index af94af01fc3e0cdb40695072dbbdc5d551de0e67..b9e0cead1b14f23b689471f545c7a43efe9a3d6f 100644
--- a/wiki/pages/design.html
+++ b/wiki/pages/design.html
@@ -9,17 +9,17 @@
<div class="col">
<h1>Our goal</h1>
- <p>Cellulopolis aims to optimize the production of bacterial cellulose (BC) by bacteria of the genus Komagataeibacter, through two main approaches: strain domestication and its gene editing, and the adoption of alternative culture media. The laboratory arm of the project is subdivided into cultivation in alternative media, establishing conditions for genetic manipulation, developing a toolkit for <i>Komagataeibacter rhaeticus</i> AF1 engineering, producing BC in defined shapes, and testing BC as a scaffold for tissue culture.</p>
+ <p>Cellulopolis aims to optimize the production of bacterial cellulose (BC) by bacteria of the genus <i>Komagataeibacter</i>, through two main approaches: strain domestication and its gene editing, and the adoption of alternative culture media. The laboratory arm of the project is subdivided into cultivation in alternative media, establishing conditions for genetic manipulation, developing a toolkit for <i>Komagataeibacter rhaeticus</i> AF1 engineering, producing BC in defined shapes, and testing BC as a scaffold for tissue culture.</p>
<p>Our design was focused on planning, building (partial) and testing (partial) novel parts and strains. To achieve engineering success utilizing a novel chassis, our main challenge was to construct a GoldenGate compatible toolkit, composed of promoters with different strengths, RBS, and terminator, in addition to level 1 and 2 backbones with different origins of replication, making its manipulation available by other research groups. From these parts, we propose a solution to increase the efficiency in the production of cellulose, from the modulation of the operon of cellulose production of the species, through the adoption of a promoter inducible by light. We also evaluated different compositions of the culture medium used, proposing the adoption of more sustainable and economically viable alternatives for the growth of the strain.</p>
<h1>Chassis selection</h1>
- <p>From the inception of the team Unicamp_Brazil, the goal was to contribute towards a more sustainable planet. Hence, when we decided to optimize BC production from waste available in large quantities in our region, we contacted BioPolMat (UNIARA), BioSmart, and HB consortium, which cultivates BC on agroindustry residues and were supplied with <i>K. rhaeticus</i> AF1. To the best of our knowledge, the genome of <i>K. rhaeticus</i> had never been edited, thus presenting a potential engineering challenge. However, the benefits of starting from a strain adapted to growth in our substrate of choice outweighed the drawbacks. As a backup strategy, we worked in parallel with one more Komagataeibacter strain, namely <i>K. medellinensis</i> ID13488.</p>
+ <p>From the inception of the team Unicamp_Brazil, the goal was to contribute towards a more sustainable planet. Hence, when we decided to optimize BC production from waste available in large quantities in our region, we contacted BioPolMat (UNIARA), BioSmart, and HB consortium, which cultivates BC on agroindustry residues and were supplied with <i>K. rhaeticus</i> AF1. To the best of our knowledge, the genome of <i>K. rhaeticus</i> had never been edited, thus presenting a potential engineering challenge. However, the benefits of starting from a strain adapted to growth in our substrate of choice outweighed the drawbacks. As a backup strategy, we worked in parallel with one more <i>Komagataeibacter</i> strain, namely <i>K. medellinensis</i> ID13488.</p>
<h1>Design strategy</h1>
- <p>Predictions from the flux balance analysis we performed using the <i>Komagataeibacter xylinus</i> genome-scale model (Rezazadeh et al, 2020), indicate that bacterial cellulose (BC) production competes with biomass accumulation, hence redirecting Komagataeibacter´s metabolism towards BC production should have detrimental effects on biomass accumulation. This negative growth pressure would lead to the selection of mutants with defects in BC-producing enzymes, as previously shown by Hur et al., 2020. Thus, we designed a bimodal strategy whereby we will engineer the bacterial cellulose biosynthetic gene cluster (bcs) to shut down BC production during biomass accumulation and induce BC production once desired.</p>
+ <p>Predictions from the flux balance analysis we performed using the <i>Komagataeibacter xylinus</i> genome-scale model (Rezazadeh et al, 2020), indicate that bacterial cellulose (BC) production competes with biomass accumulation, hence redirecting <i>Komagataeibacter</i>´s metabolism towards BC production should have detrimental effects on biomass accumulation. This negative growth pressure would lead to the selection of mutants with defects in BC-producing enzymes, as previously shown by Hur et al., 2020. Thus, we designed a bimodal strategy whereby we will engineer the bacterial cellulose biosynthetic gene cluster (bcs) to shut down BC production during biomass accumulation and induce BC production once desired.</p>
<p>The entire premise of our project is to minimize BC production costs, hence the use of a low-cost inducer is imperative. This requires the use of inducible promotors which can be switched on once the desired cell density is achieved. Literature searches revealed only a couple of inducible promoters functional in <i>K. rhaeticus</i>, such as LuxR (activated by acyl homoserine lactone - AHL) (Goosens et al., 2021). In spite of the good characterization, LuxR is not an option for scalable induction due to the cost of AHL. Fortunately we came across a paper describing a single component light responsive system for control of gene expression in <i>E. coli</i>: the LexRO (Li et al.,2020) . Under dark conditions LexRO dimers interact with an operator region upstream of the RBS, inhibiting transcription. When exposed to blue light, LexRO proteins dissociate and detach from the DNA, allowing transcription.</p>
<p>Based on Florea et al (Florea et al., 2016), we synthesized promotors with high (TUp_a), medium (TUp_b) and low (TUp_c) strengths. These were coupled to optimized RBS (Hur et al, 2020) (TU_RBS), and strong terminator (TU_term) (Florea et al 2016).</p>
<p><i>Komagataeibacter rhaeticus</i> AF1 genome is highly GC rich, therefore we ordered gene synthesis to represent this codon preference. Unfortunately our custom parts arrived only a couple of weeks prior to the wiki freeze so the composite parts required for validation of the LexRO in <i>K. rhaeticus</i> were not ready on time. Our plan is to assemble 3 plasmids encoding multiple transcriptional units each: LexRO under the control of strong, medium and weak promotors and lex operator controling the expression of a purple reporter (for details and references, see parts section). These will be transformed into <i>K. rhaeticus</i> and will be cultivated in dark (foil covered flask or plate) or light (custom constructed light box - for details see hardware session) conditions. Colour profile of <i>K. rhaeticus</i> cells should indicate the levels if LexRO expression efficient in inhibiting transcription under dark condition (white cells) and allowing transcription under blue light (purple cells).</p>
@@ -38,16 +38,16 @@
<p>The genomes of <i>K. rhaeticus</i> AF1 and <i><i>K. medellinensis</i></i> ID13488 have been sequenced (Hernández-Arriaga et al. 2019; Santos et al. 2019), however, to the best of our knowledge neither strain has been previously manipulated. Hence, to permit engineering and optimization of BC production, we began by evaluating the susceptibility of each strain to the 4 classic antibiotics: ampicillin, kanamycin, spectinomycin and chloramphenicol.</p>
<h1>Build</h1>
- <h2>Komagataeibacter compatible plasmids</h2>
+ <h2><i>Komagataeibacter</i> compatible plasmids</h2>
- <p>According to the literature, standard replication origins do not work efficiently in Komagataeibacter, therefore we selected backbones with the broad spectrum origins RK2 (BBa_J428346; BBa_J428366), pBBR1(BBa_J428347; BBa_J428367) or RSF1010 (BBa_J428349; BBa_J428369) for further work (Fricke et al 2021).</p>
+ <p>According to the literature, standard replication origins do not work efficiently in <i>Komagataeibacter</i>, therefore we selected backbones with the broad spectrum origins RK2 (BBa_J428346; BBa_J428366), pBBR1(BBa_J428347; BBa_J428367) or RSF1010 (BBa_J428349; BBa_J428369) for further work (Fricke et al 2021).</p>
<p>To evaluate the ameneability of <i>K. rhaeticus</i> AF1 and <i>K. medellinensis</i> ID13488 to genetic manipulations, we transformed both strains by electroporation with BBa_J428346, BBa_J428347 and BBa_J428349, as suggested by Florea, 2016. Initially we struggled with poor transformation efficiency and contamination. After several rounds of frustrated transformation attempts, using a new optimized protocol we acchieved transformation success.</p>
- <p>Unfortunately the parts suplied with the iGEM distribution kit had few selection markers, with the only level 2 backbones with broad spectrum origins harbouring spectinomycin resistance cassette, which, to the best of our knowledge, has not been shown to be effective in Komagataeibacter. Therefore we deviced SapI Golden Gate based strategy to replace the KanR marker from level 1 plasmid and replace the SpecR marker from level 2 plasmids with AmpR and CmR. Plasmid backbones BBa_J428346, BBa_J428347, BBa_J428349 , BBa_J428366, BBa_J428367, and BBa_J428369 were amplified with primers JUMP-mF and JUMP-mR and antibiotic resistance cassettes were amplified with AmpR_F, AmpR_R, CmR_F and CmR_R using plasmids BBa_J428385 or BBa_J428357 as templates (Figure y).</p>
+ <p>Unfortunately the parts suplied with the iGEM distribution kit had few selection markers, with the only level 2 backbones with broad spectrum origins harbouring spectinomycin resistance cassette, which, to the best of our knowledge, has not been shown to be effective in <i>Komagataeibacter</i>. Therefore we deviced SapI Golden Gate based strategy to replace the KanR marker from level 1 plasmid and replace the SpecR marker from level 2 plasmids with AmpR and CmR. Plasmid backbones BBa_J428346, BBa_J428347, BBa_J428349 , BBa_J428366, BBa_J428367, and BBa_J428369 were amplified with primers JUMP-mF and JUMP-mR and antibiotic resistance cassettes were amplified with AmpR_F, AmpR_R, CmR_F and CmR_R using plasmids BBa_J428385 or BBa_J428357 as templates (Figure y).</p>
<h2>Level 2 backbone adaptors</h2>
- <p>To allow the assembly of multiple transcriptional units we are modifying the cloning site of level 2 vectors with replication origins functional in Komagataeibacter, by replacing the region between BsaI sites of plasmids derived from BBa_J428366 and BBa_J428367 with an adapter suitable for simultaneous cloning 4 transcriptional units by GoldenGate with SapI. This is done by amplifying the red fluorescent cassettefrom BBa_J04452, and cloning by GoldenGate with BsaI digestion into BBa_K4435304, BBa_K4435305, BBa_K4435311 and BBa_K4435312, generating plasmids BBa_K4435306, BBa_K4435307, BBa_K4435313 and BBa_K4435314.</p>
+ <p>To allow the assembly of multiple transcriptional units we are modifying the cloning site of level 2 vectors with replication origins functional in <i>Komagataeibacter</i>, by replacing the region between BsaI sites of plasmids derived from BBa_J428366 and BBa_J428367 with an adapter suitable for simultaneous cloning 4 transcriptional units by GoldenGate with SapI. This is done by amplifying the red fluorescent cassettefrom BBa_J04452, and cloning by GoldenGate with BsaI digestion into BBa_K4435304, BBa_K4435305, BBa_K4435311 and BBa_K4435312, generating plasmids BBa_K4435306, BBa_K4435307, BBa_K4435313 and BBa_K4435314.</p>
<p>The replacement of the green fluorescent markers from plasmids BBa_K4435304, BBa_K4435305, BBa_K4435311 and BBa_K4435312 with a red fluorescent marker flanked by the TU adapters allows the visual selection of sucessfull constructs.</p>
@@ -59,7 +59,7 @@
<p>Genes encoding BC-producing enzymes and BC transporters are organized into different bcs operons (​​Orlovska et al, 2021). The type I cellulose synthase operon (bcsI) comprises four genes, bcsA, bcsB, bcsC and bcsD, flanked by the cellulose synthesis modulators bcsZ, bcsH and bglX (Römling and Galperin 2015). We decided to control BC production by introducing a LexRO binding region upstream of cmcAx (upstream construct) and directly upstream of bcsA (mid construct), the cellulose synthase encoding gene.</p>
<figure>
<img class="pages-img" src="https://static.igem.wiki/teams/4435/wiki/design/bcs-operon.svg">
- <figcaption>Figure . Schematic illustration of Komagataeibacter´s bcs operon indicating engineering sites.</figcaption>
+ <figcaption>Figure . Schematic illustration of <i>Komagataeibacter</i>´s bcs operon indicating engineering sites.</figcaption>
</figure>
<h2>Genomic integration</h2>
@@ -90,7 +90,7 @@
<p>TU4 type parts encoding the synthetic LexRO binding site with RBS, followed by either cmcax or bcsA (1 kb 3´homology) are being constructed by Golden gate assembly of BBa_K4435016 with the PCR products of amplifications from genomic DNA from either <i>K. rhaeticus</i> AF1 or <i>K. medellinensis</i>.</p>
- <p>Recombination of FRTs require the expression of FLP recombinases, therefore we design a strategy to express either yeast FLP (BBF10K_000210) or newly designed enhanced FLP, called FLPe, which was codon optimized for Komagataeibacter (BBa_K4435002). We are cloning the recombinases under the control of strong promotors (BBa_K4435105; BBa_K4435106) for transformation into Komagataibacter once correct genomic integration of the bcs locus cassettes is verified.</p>
+ <p>Recombination of FRTs require the expression of FLP recombinases, therefore we design a strategy to express either yeast FLP (BBF10K_000210) or newly designed enhanced FLP, called FLPe, which was codon optimized for <i>Komagataeibacter</i> (BBa_K4435002). We are cloning the recombinases under the control of strong promotors (BBa_K4435105; BBa_K4435106) for transformation into <i>Komagataeibacter</i> once correct genomic integration of the bcs locus cassettes is verified.</p>
<h2>Overexpression of further genes encoding enzymes involved in BC synthesis</h2>
@@ -102,7 +102,7 @@
</figcaption>
</figure>
- <p>It has been postulated that expression levels of GK, galU, pgm, ndp and/or dgc could redirect glucose towards cellulose production, increasing final yields. To identify the limiting enzyme in the pathway and device a strategy for maximum BC production, we designed primers and amplified genes encoding GK, galU, pgm and ndp from genomic DNA of <i>K. rhaeticus</i> AF1 and <i>K. medellinensis</i>. These are being cloned into BBa_J428381, BBa_J428382, BBa_J428383, and BBa_J428384, under the control of the strong constitutive promotor BBa_K4435012, RBS BBa_K4435015 and terminator BBa_K4435017 (level 1 composite parts BBa_K4435120 to BBa_K4435127). Parts BBa_K4435120 to BBa_K4435123 will be combined into BBa_K4435313 to produce BBa_K4435217 and parts BBa_K4435124 to BBa_K4435127 will be combined into BBa_K4435313 to produce BBa_K4435218. BBa_K4435217 and BBa_K4435218 will be transformed into Komagataeibacter and BC production quantified. If there is an increase in cellulose production, we will test individual parts to determine the limiting enzyme in the pathway. All these used primers and amplified genes are disponible at parts page (link).</p>
+ <p>It has been postulated that expression levels of GK, galU, pgm, ndp and/or dgc could redirect glucose towards cellulose production, increasing final yields. To identify the limiting enzyme in the pathway and device a strategy for maximum BC production, we designed primers and amplified genes encoding GK, galU, pgm and ndp from genomic DNA of <i>K. rhaeticus</i> AF1 and <i>K. medellinensis</i>. These are being cloned into BBa_J428381, BBa_J428382, BBa_J428383, and BBa_J428384, under the control of the strong constitutive promotor BBa_K4435012, RBS BBa_K4435015 and terminator BBa_K4435017 (level 1 composite parts BBa_K4435120 to BBa_K4435127). Parts BBa_K4435120 to BBa_K4435123 will be combined into BBa_K4435313 to produce BBa_K4435217 and parts BBa_K4435124 to BBa_K4435127 will be combined into BBa_K4435313 to produce BBa_K4435218. BBa_K4435217 and BBa_K4435218 will be transformed into <i>Komagataeibacter</i> and BC production quantified. If there is an increase in cellulose production, we will test individual parts to determine the limiting enzyme in the pathway. All these used primers and amplified genes are disponible at parts page (link).</p>
<table class="page-parts">
<tr><th>Primers F</th><th>Sequence</th><th>Primer R</th><th>Sequence</th></tr>
@@ -113,7 +113,7 @@
</table>
- <p>C-di-GMP is an essential activator of bcsA, hence we ordered with IDT the gene encoding the enzyme dgc, responsible for c-di-GMP synthesis. This is being cloned into the pSB1C3C backbone (new basic part BBa_K4435003). BBa_K4435003 will be cloned into BBa_J428384, with BBa_K4435012, BBa_K4435015 and BBa_K4435022 (new composite part BBa_K4435128). This will be combined with level 1 parts type BBa_J428381, BBa_J428382 and BBa_J428383 in BBa_K4435306. Level 2 composite parts will be transformed into Komagataeibacter for BC production.</p>
+ <p>C-di-GMP is an essential activator of bcsA, hence we ordered with IDT the gene encoding the enzyme dgc, responsible for c-di-GMP synthesis. This is being cloned into the pSB1C3C backbone (new basic part BBa_K4435003). BBa_K4435003 will be cloned into BBa_J428384, with BBa_K4435012, BBa_K4435015 and BBa_K4435022 (new composite part BBa_K4435128). This will be combined with level 1 parts type BBa_J428381, BBa_J428382 and BBa_J428383 in BBa_K4435306. Level 2 composite parts will be transformed into <i>Komagataeibacter</i> for BC production.</p>
</div>
</div>
diff --git a/wiki/pages/engineering.html b/wiki/pages/engineering.html
index cd25dbeac9d08dc7cf42a2942d916dc52604a9be..2db6356740f3586122b9ff384aa1670f08ed884d 100644
--- a/wiki/pages/engineering.html
+++ b/wiki/pages/engineering.html
@@ -117,7 +117,7 @@
<p>Our engineering started with the conception of the project, which focused on dealing with environmental problems and providing a positive outcome for society. We began constructing it from the information we collected with research on waste management and visits to recycling centers. This culminated with the idea of working with an enzyme capable of degrading polystyrene, the main component of styrofoam, which has almost no destination other than landfills, because of the lack of profitability in its recycling process. Thus, we began to run the tests for pre-treatment of the material and encountered some barriers due to the approach’s inefficiency, such as a negative environmental impact when pollution due to transport was accounted for. With this scenario, we learned that our first idea could be unfeasible and re-set the design project design for the production of BC from agroindustrial residues, a project that actually shows consistent results regarding positive social and environmental impact.</p>
<h1>Define antibiotic concentrations and establish electroporation protocols</h1>
- <p>An important step in engineering our chassis is the transformation of <i>Komagataeibacter</i> with the plasmids of interest. We planned this step from extensive literature research and protocol analysis in order to tailor efficient instructions for the strain of interest. We tested the original transformation protocols published for <i>K. rhaeticus</i> iGEM, using our Komagataeibacter rhaeticus AF1 strain. Unfortunately, our first attempts were not successful, since it was not possible to observe the growth of transformed strains on the antibiotic plates. This scenario led us to question the antibiotic concentrations adopted in the published protocol and perform experiments to test the resistance of <i>K. rhaeticus</i> AF1 to various concentrations of the antibiotics Kanamycin, Ampicilin and Chloramphenicol. With the results of this experiment, we interpreted the bacterial response to different antibiotics and learned to adapt the protocol efficiently. From there, we rethought the design for the transformation, establishing optimized protocols for retesting, which allowed us to successfully transform <i>K. rhaeticus</i> AF1 for the first time. It is important to note that we successfully transformed <i>K. rhaeticus</i> AF1 with novel backbones constructed in this project. </p>
+ <p>An important step in engineering our chassis is the transformation of <i>Komagataeibacter</i> with the plasmids of interest. We planned this step from extensive literature research and protocol analysis in order to tailor efficient instructions for the strain of interest. We tested the original transformation protocols published for <i>K. rhaeticus</i> iGEM, using our <i>Komagataeibacter</i> rhaeticus AF1 strain. Unfortunately, our first attempts were not successful, since it was not possible to observe the growth of transformed strains on the antibiotic plates. This scenario led us to question the antibiotic concentrations adopted in the published protocol and perform experiments to test the resistance of <i>K. rhaeticus</i> AF1 to various concentrations of the antibiotics Kanamycin, Ampicilin and Chloramphenicol. With the results of this experiment, we interpreted the bacterial response to different antibiotics and learned to adapt the protocol efficiently. From there, we rethought the design for the transformation, establishing optimized protocols for retesting, which allowed us to successfully transform <i>K. rhaeticus</i> AF1 for the first time. It is important to note that we successfully transformed <i>K. rhaeticus</i> AF1 with novel backbones constructed in this project. </p>
<h1>Design new level 1 and level 2 backbones with selection markers compatible with <i>Komagataeibacter</i></h1>
diff --git a/wiki/pages/hardware.html b/wiki/pages/hardware.html
index b4696ca34f10cea8a7807a5719f026534bcf4fbe..d37c827095d75289769b414cb203dce8c659c535 100644
--- a/wiki/pages/hardware.html
+++ b/wiki/pages/hardware.html
@@ -10,7 +10,7 @@
-<p>Bacterial cellulose (BC) is a versatile material that could potentially be used as a scaffold for tissue engineering. Therefore, our team is optimizing Komagataeibacter for the production of BC and testing the development of molds that would allow the growth of BC sheets in the correct format for tissue culture and subsequent 3D organ assembly. To that end, we converted the surface of a complex object into a 2D shape, printed the perimeter as a “cookie cutterâ€, and used it as a template for bacterial cellulose sheet production.</p>
+<p>Bacterial cellulose (BC) is a versatile material that could potentially be used as a scaffold for tissue engineering. Therefore, our team is optimizing <i>Komagataeibacter</i> for the production of BC and testing the development of molds that would allow the growth of BC sheets in the correct format for tissue culture and subsequent 3D organ assembly. To that end, we converted the surface of a complex object into a 2D shape, printed the perimeter as a “cookie cutterâ€, and used it as a template for bacterial cellulose sheet production.</p>
<p>Unfolding the shape of a sphere into a 2D object</p>
@@ -34,15 +34,15 @@
<h2> Bioreactors</h2>
-<p>The project design aims to engineer strains with a bimodal growth pattern, divided in biomass accumulation stage and Bc production stage. To achieve this we are engineering a K. rhaeticus AF1 where cellulose synthase genes are off under dark and expressed by the use of blue light. Therefore, we devised a bioreactor to grow Komagataeibacter in an environment where there is no light and switch to an inducible environment when needed.</p>
+<p>The project design aims to engineer strains with a bimodal growth pattern, divided in biomass accumulation stage and Bc production stage. To achieve this we are engineering a K. rhaeticus AF1 where cellulose synthase genes are off under dark and expressed by the use of blue light. Therefore, we devised a bioreactor to grow <i>Komagataeibacter</i> in an environment where there is no light and switch to an inducible environment when needed.</p>
<h2>Growth optimization.</h2>
-<p>For growth optimization, some criteria were considered in the design of the bioreactor. As was seen in the math models for growth the Komagataeibacter is obligately aerobic, so the oxygen supply is very important. Another point discussed was the cost of construction. Fitting the bioreactor with an impeller would significantly increase the cost, therefore, to allow for mixing and oxygen supply, it was decided to build an air lift bioreactor.</p>
+<p>For growth optimization, some criteria were considered in the design of the bioreactor. As was seen in the math models for growth the <i>Komagataeibacter</i> is obligately aerobic, so the oxygen supply is very important. Another point discussed was the cost of construction. Fitting the bioreactor with an impeller would significantly increase the cost, therefore, to allow for mixing and oxygen supply, it was decided to build an air lift bioreactor.</p>
<h2>Production optimization.</h2>
-<p>We investigated different induction alternatives that would allow the expression of cellulose synthate (bcs) when we desired, however, the costs of inducers for the characterized Komagataeibacter promotors are prohibitive. Hence, we explored the scientific literature and encountered a recent paper describing a single-component light-sensitive transcriptional repressor. By using a light-sensitive promoter (LexRO) the production of BC can become cheaper. However, to induce the reaction efficiently, a bright place for the reaction is needed. </p>
+<p>We investigated different induction alternatives that would allow the expression of cellulose synthate (bcs) when we desired, however, the costs of inducers for the characterized <i>Komagataeibacter</i> promotors are prohibitive. Hence, we explored the scientific literature and encountered a recent paper describing a single-component light-sensitive transcriptional repressor. By using a light-sensitive promoter (LexRO) the production of BC can become cheaper. However, to induce the reaction efficiently, a bright place for the reaction is needed. </p>
<p>To unite the two needs the team developed two pieces of hardware. The first part of the growth will be done in a low-cost reactor. The first part of the growth will be done in a low-cost reactor, made out of common laboratory materials. For the fermentation vessel, a 1-liter reagent bottle was chosen. All inlets were located in the lid, following the design of commercial bioreactors. For the air inlet, a small aquarium compressor was used (4 L/min). For prototyping purposes, this reactor was tested with E. coli, due to its fast growth rate. </p>
diff --git a/wiki/pages/implementation.html b/wiki/pages/implementation.html
index 99a0f314f713c603e23d6a67b4d67d57cc7cbffe..8cd1fb18d2f02db693f3e420b895fee58a84b155 100644
--- a/wiki/pages/implementation.html
+++ b/wiki/pages/implementation.html
@@ -36,7 +36,7 @@
<div class="col">
<h2>Small scale implementation challenges</h2>
<img src="https://static.igem.wiki/teams/4435/wiki/proposed-implem/imagen2-png.png" class="figure-left">
- <p>As challenges, we can mention the great handling difficulty that the Komagataeibacter bacterium provides. This organism takes 3 days to grow, which delays the process a little depending on demand and urgency. Komagataeibacter is a reasonably expensive bacterium to maintain, because its culture medium, HS (Hestrin; Schramm, 1954), commonly used in laboratories, carries a large amount of glucose, being 50g, while most common media takes around 2g - 4g. It is also valid to bring the complication in automating the removal of the cellulose blanket, after its production, in addition to the wide obstacle encountered in making this whole process, of cellulose production, become continuous, for reasons of possible contamination and also the impasse encountered at the time of removal of the cellulose produced, considering that when this polymer is removed, the cell is also removed.</p>
+ <p>As challenges, we can mention the great handling difficulty that the <i>Komagataeibacter</i> bacterium provides. This organism takes 3 days to grow, which delays the process a little depending on demand and urgency. <i>Komagataeibacter</i> is a reasonably expensive bacterium to maintain, because its culture medium, HS (Hestrin; Schramm, 1954), commonly used in laboratories, carries a large amount of glucose, being 50g, while most common media takes around 2g - 4g. It is also valid to bring the complication in automating the removal of the cellulose blanket, after its production, in addition to the wide obstacle encountered in making this whole process, of cellulose production, become continuous, for reasons of possible contamination and also the impasse encountered at the time of removal of the cellulose produced, considering that when this polymer is removed, the cell is also removed.</p>
<img src="https://static.igem.wiki/teams/4435/wiki/proposed-implem/imagen3-celulosa.png" class="figure-left">
</div>
</div>
diff --git a/wiki/pages/model.html b/wiki/pages/model.html
index 06d8f8a3ca4adbbf01bcf8f354ce417c8fba0c3d..aa436b2122a5b9ba4c5719452741cab9ac0ba764 100644
--- a/wiki/pages/model.html
+++ b/wiki/pages/model.html
@@ -17,7 +17,7 @@
<p>A genomic-scale metabolic model (GEM) contains all or most the metabolites and reactions of a microorganism. It can be used to predict the behavior of the microorganism under certain conditions and to understand the main reactions involved in the synthesis of the product of interest. It can also be used to understand the effect that different gene knockouts can have on the cell.</p>
<p>Building a metabolic network is a long and arduous process, for it involves the reconstruction of a complete reaction-by-reaction network. However, due to the joint efforts of several researchers, currently, there are collections with many networks and subnetworks available.</p>
<p>These initial steps create a model representing all the enzymes encoded by the organism's genome. With these enzymes and the use of collections of enzymatic reactions, it is possible to define the metabolites that these enzymes act on. Once the connection network between the metabolites is defined, these data need to be organized so that they can be analyzed mathematically. After having identified all the metabolites and the reactions, the latter needs to be balanced. With these data, a stoichiometric matrix of all the metabolites present in the model is constructed.</p>
- <p>The model that was used in the present project was published by Rezazadeh et al. (2020) for Komagataeibacter xylinus, a species closely related to K. rhaeticus, allowing us to use the same model to gain insights into the metabolism of our strain.</p>
+ <p>The model that was used in the present project was published by Rezazadeh et al. (2020) for <i>Komagataeibacter xylinus</i>, a species closely related to K. rhaeticus, allowing us to use the same model to gain insights into the metabolism of our strain.</p>
<h1>Methods for the solution of Genomic-scale Metabolic Models</h1>
<p>After the model construction, it is necessary to check its consistency. The verification can be done manually for small models, but in the case of the K. xylinus’ model, it is unfeasible, as it has a total of 865 metabolites and 918 reactions. The way to do this check automatically is with the use of the software. We used the MEMOTE app, which is a free and open-source web application.</p>
@@ -66,7 +66,7 @@
<h1>Medium conditions limitations</h1>
<p>Using the GEM described above, it is possible to change the uptake conditions and use this to understand the minimum requirements for the culture media.</p>
- <p>In collaboration with the Uioslo-Norway team, which goal is to use Komagataeibacter in co-culture to produce a copolymer containing cellulose and chitin. During the production of chitin, it is necessary to use nitrogen. So, to better understand the cultivation requirements, different cultivation conditions are simulated to suggest how much NH4 the Komagataeibacter needs for its development. We determined minimal NH4 for growth as well as oxygen requirements of the obligate aerobic bacteria. Thus, the model was used to test different media conditions changing the NH4 and O2 available to determine the minimum for cell growth and the minimum for cellulose production. Cellular uptake can be seen in the table below and in Figure 4.</p>
+ <p>In collaboration with the Uioslo-Norway team, which goal is to use <i>Komagataeibacter</i> in co-culture to produce a copolymer containing cellulose and chitin. During the production of chitin, it is necessary to use nitrogen. So, to better understand the cultivation requirements, different cultivation conditions are simulated to suggest how much NH4 the <i>Komagataeibacter</i> needs for its development. We determined minimal NH4 for growth as well as oxygen requirements of the obligate aerobic bacteria. Thus, the model was used to test different media conditions changing the NH4 and O2 available to determine the minimum for cell growth and the minimum for cellulose production. Cellular uptake can be seen in the table below and in Figure 4.</p>
<img class="model-page-tbl" src="https://static.igem.wiki/teams/4435/wiki/pages/modeling/table02.svg">
@@ -85,14 +85,14 @@
<figcaption>Figure 5. FO - Biomass (upper panel) and FO cellulose (lower panel) showing enzymes that could impact biomass accumulation when the corresponding genes are overexpressed.</figcaption>
</figure>
- <p>Figure 5 shows changes in the ranges of various reactions within the microorganism. They illustrate that the flexibility of some reactions is affected when there is a change in the objective function. These reactions that had their ranges reduced are essential for the production of cellulose in Komagataibacter.</p>
+ <p>Figure 5 shows changes in the ranges of various reactions within the microorganism. They illustrate that the flexibility of some reactions is affected when there is a change in the objective function. These reactions that had their ranges reduced are essential for the production of cellulose in <i>Komagataeibacter</i>.</p>
<h1>Yields of cellulose in culture</h1>
<p>Cellular growth has complex effects on cellulose yields, making modeling quite difficult. However, there are simplified mathematical models that can be used to get good insights into growth and BC production. One of the main parameters used is the metabolic yield coefficients which are the ratio between two variables of interest. eg biomass (x) and substrate(s):</p>
<img class="model-page-eq" src="https://static.igem.wiki/teams/4435/wiki/pages/modeling/equation01.svg">
- <p>The Yxs coefficient indicates how much of the substrate is converted into biomass. In the case of Komagataeibacter xylinus, 1.7 to 2.4% of the substrate is used for biomass.</p>
+ <p>The Yxs coefficient indicates how much of the substrate is converted into biomass. In the case of <i>Komagataeibacter xylinus</i>, 1.7 to 2.4% of the substrate is used for biomass.</p>
<img class="model-page-eq" src="https://static.igem.wiki/teams/4435/wiki/pages/modeling/equation02.svg">
@@ -100,7 +100,7 @@
<img class="model-page-eq" src="https://static.igem.wiki/teams/4435/wiki/pages/modeling/equation03.svg">
- <p>The coefficient Ypx indicates the product of interest generated for each g of biomass. In the case of Komagataeibacter xylinus, for 1 g of biomass 10g of cellulose is producted.</p>
+ <p>The coefficient Ypx indicates the product of interest generated for each g of biomass. In the case of <i>Komagataeibacter xylinus</i>, for 1 g of biomass 10g of cellulose is producted.</p>
<h1>Growth kinetics and production</h1>
<p>In this work, the behavior was modeled only in a batch system. I was assuming perfect mixing and homogeneous conditions.</p>
@@ -123,7 +123,7 @@
<img class="model-page-eq" src="https://static.igem.wiki/teams/4435/wiki/pages/modeling/equation07.svg">
<h2>Product Formation</h2>
- <p>Wild-type Komagataeibacter has indirect product formation, but for modeling purposes, it will be considered associated with mixed-mode energy metabolism.</p>
+ <p>Wild-type <i>Komagataeibacter</i> has indirect product formation, but for modeling purposes, it will be considered associated with mixed-mode energy metabolism.</p>
<img class="model-page-eq" src="https://static.igem.wiki/teams/4435/wiki/pages/modeling/equation08.svg">
@@ -204,15 +204,15 @@
<div class="references">
<h1>References</h1>
- <p>1. 	 Rezazadeh, M., Babaeipour, V. & Motamedian, E. Reconstruction, verification and in-silico analysis of a genome-scale metabolic model of bacterial cellulose producing Komagataeibacter xylinus. Bioprocess Biosyst. Eng. 43, 1017–1026 (2020).</p>
+ <p>1. 	 Rezazadeh, M., Babaeipour, V. & Motamedian, E. Reconstruction, verification and in-silico analysis of a genome-scale metabolic model of bacterial cellulose producing <i>Komagataeibacter xylinus</i>. Bioprocess Biosyst. Eng. 43, 1017–1026 (2020).</p>
<p>2. 	 Orth, J. D., Thiele, I. & Palsson, B. O. What is flux balance analysis? Nat. Biotechnol. 28, 245–248 (2010).</p>
<p>3. 	 Lieven, C. et al. MEMOTE for standardized genome-scale metabolic model testing. Nat. Biotechnol. 38, 272–276 (2020).</p>
<p>4. 	 Zhong, C. et al. Revealing differences in metabolic flux distributions between a mutant strain and its parent strain Gluconacetobacter xylinus CGMCC 2955. PLoS One 9, (2014).</p>
<p>5. 	 Jenior, M. L., Moutinho, T. J., Dougherty, B. V. & Papin, J. A. Transcriptome-guided parsimonious flux analysis improves predictions with metabolic networks in complex environments. PLoS Comput. Biol. 16, (2020).</p>
<p>6. 	 Gudmundsson, S. & Thiele, I. Computationally efficient flux variability analysis. BMC Bioinformatics 11, 2–4 (2010).</p>
- <p>7. 	 Hur, D. H. et al. Enhanced production of bacterial cellulose in komagataeibacter xylinus via tuning of biosynthesis genes with synthetic RBS. J. Microbiol. Biotechnol. 30, 1430–1435 (2020).</p>
+ <p>7. 	 Hur, D. H. et al. Enhanced production of bacterial cellulose in <i>Komagataeibacter xylinus</i> via tuning of biosynthesis genes with synthetic RBS. J. Microbiol. Biotechnol. 30, 1430–1435 (2020).</p>
<p>8. 	 Bioprocess Engineering Principles (Second Edition). in Bioprocess Engineering Principles (Second Edition) (ed. Doran, P. M.) ii (Academic Press, 2013). doi:https://doi.org/10.1016/B978-0-12-220851-5.00017-4.</p>
- <p>9. 	 Reiniati, I., Hrymak, A. N. & Margaritis, A. Kinetics of cell growth and crystalline nanocellulose production by Komagataeibacter xylinus. Biochem. Eng. J. 127, 21–31 (2017).</p>
+ <p>9. 	 Reiniati, I., Hrymak, A. N. & Margaritis, A. Kinetics of cell growth and crystalline nanocellulose production by <i>Komagataeibacter xylinus</i>. Biochem. Eng. J. 127, 21–31 (2017).</p>
<p>10. 	 Palsson, B. (2015). Systems Biology: Constraint-based Reconstruction and Analysis. Cambridge: Cambridge University Press. doi:10.1017/CBO9781139854610</p>
</div>
diff --git a/wiki/pages/notebook.html b/wiki/pages/notebook.html
index 57598eb77cbc2d37e042e55771b6ce363507d40a..76fbe92669b1a9883106cfe83bdfd6800669e3d8 100644
--- a/wiki/pages/notebook.html
+++ b/wiki/pages/notebook.html
@@ -198,7 +198,7 @@ D4, F6, D8 (AmpR)
<p>Grow K. rhaeticus AF1 and K. medellinensis in different media to quantify BC production</p>
<h1>September 2022: third week </h1>
-<p>Minipreps of toolkit parts for new construct design (low yield; shaker kept at 30oC due to Komagataibacter cultures; probably low yield due to poor E.coli growth; solution: incubate for longer and with larger volume in larger tubes)</p>
+<p>Minipreps of toolkit parts for new construct design (low yield; shaker kept at 30oC due to <i>Komagataeibacter</i> cultures; probably low yield due to poor E.coli growth; solution: incubate for longer and with larger volume in larger tubes)</p>
<p>Weight BC wet sheets:
AF1- wet sheet 1,113 g
@@ -285,7 +285,7 @@ hold at 4oC</p>
<p>Golden Gate LexRO and FLPe into plasmid 13p (iGEM backbone) – fail</p>
<p>(26/09)</p>
-<p>Measure OD of Komagataeibacter cultures to adjust OD:
+<p>Measure OD of <i>Komagataeibacter</i> cultures to adjust OD:
<p><i>K. medellinensis</i>: 0,354
<i>K. rhaeticus</i> AF1: 0,605</p>
@@ -349,7 +349,7 @@ hold at 4oC</p>
<p>TU_RBS</p>
<p>(Did not work)</p>
-<p>PCR of Komagataeibacter genes with new IDT primers</p>
+<p>PCR of <i>Komagataeibacter</i> genes with new IDT primers</p>
<p>galU</p>
<p>pgm (1,7 kb)</p>
<p>ndp (0,5 kb)</p>
diff --git a/wiki/pages/parts.html b/wiki/pages/parts.html
index 282939a8e048e0861f4c2199b639e9f479171d4a..cff1266d386085280746d4a6e1c311857086c42e 100644
--- a/wiki/pages/parts.html
+++ b/wiki/pages/parts.html
@@ -53,7 +53,7 @@
<h2>Construction of new broad species backbones</h2>
-<p>According to multiple scientific papers, standard E. coli compatible replication origins are not functional in Komagataeibacter. Unusual chassis frequently require plasmids with broad species origins such as RK2, pBBR1 or RSF1010 (present in iGEM plasmids BBa_J428346, BBa_J428347, BBa_J428349, BBa_J428366, BBa_J428367 and BBa_J428369). Plasmids BBa_J428346, BBa_J428347, BBa_J428349 also encode the KanR marker and whereas BBa_J428366, BBa_J428367 and BBa_J428369 encode SpecR (the latter of which has not been proven effective in Komagataeibacter). Hence, the level 2 vectors supplied with the distribution kit were not compatible with our chassis. To overcome this, we designed primers JUMP_mF and JUMP_mR to amplify BBa_J428346, BBa_J428347, BBa_J428349, BBa_J428366, BBa_J428367 and BBa_J428369 excluding the KanR and SpecR selection markers. In parallel, we designed primers AmpR_F and AmpR_R to amplify the Ampicillin resistance cassette from BBa_J428385 and primers CmR_F and CmR_R to amplify the chloramphenicol resistance cassette from BBa_J428357.</p>
+<p>According to multiple scientific papers, standard E. coli compatible replication origins are not functional in <i>Komagataeibacter</i>. Unusual chassis frequently require plasmids with broad species origins such as RK2, pBBR1 or RSF1010 (present in iGEM plasmids BBa_J428346, BBa_J428347, BBa_J428349, BBa_J428366, BBa_J428367 and BBa_J428369). Plasmids BBa_J428346, BBa_J428347, BBa_J428349 also encode the KanR marker and whereas BBa_J428366, BBa_J428367 and BBa_J428369 encode SpecR (the latter of which has not been proven effective in <i>Komagataeibacter</i>). Hence, the level 2 vectors supplied with the distribution kit were not compatible with our chassis. To overcome this, we designed primers JUMP_mF and JUMP_mR to amplify BBa_J428346, BBa_J428347, BBa_J428349, BBa_J428366, BBa_J428367 and BBa_J428369 excluding the KanR and SpecR selection markers. In parallel, we designed primers AmpR_F and AmpR_R to amplify the Ampicillin resistance cassette from BBa_J428385 and primers CmR_F and CmR_R to amplify the chloramphenicol resistance cassette from BBa_J428357.</p>
<figure>
<img class="pages-img" src="https://static.igem.wiki/teams/4435/wiki/pages/parts/parts-1.png">
@@ -113,7 +113,7 @@
<tr><td>BBa_K4435314</td><td>add ODD 4 TUs adapter to BBa_K4435312</td><td>6M to CmR_red</td><td></td></tr>
</table>
-<p>In addition to the novel backbones we also constructed a range of novel basic parts. The highlight of this collection are gene encoding the light-sensitive transcriptional repressor LexRO (BBa_K4435001) and its associated operon (BBa_K4435016). Unfortunately, these customs parts synthesized by IDT arrived just less than 3 weeks prior to the wiki freeze, which did not allow us time to evaluate their activity in Komagataibacter. We amplified 1 kb genomic DNA fragments from K. rhaeticus and K. medellinensis that will flank our constructs directing their integration into the bacteria genome directly upstream of either bcsZ or bcsA. We also cloned a series of promotors, RBS, terminator and genes encoding key enzymes in the BC synthesis pathway to allow their overexpression and consequently redirect the metabolic flux towards BC production. Most basic parts have been cloned into pSB1C3 plasmids and await confirmation by sequencing. </p>
+<p>In addition to the novel backbones we also constructed a range of novel basic parts. The highlight of this collection are gene encoding the light-sensitive transcriptional repressor LexRO (BBa_K4435001) and its associated operon (BBa_K4435016). Unfortunately, these customs parts synthesized by IDT arrived just less than 3 weeks prior to the wiki freeze, which did not allow us time to evaluate their activity in <i>Komagataeibacter</i>. We amplified 1 kb genomic DNA fragments from K. rhaeticus and K. medellinensis that will flank our constructs directing their integration into the bacteria genome directly upstream of either bcsZ or bcsA. We also cloned a series of promotors, RBS, terminator and genes encoding key enzymes in the BC synthesis pathway to allow their overexpression and consequently redirect the metabolic flux towards BC production. Most basic parts have been cloned into pSB1C3 plasmids and await confirmation by sequencing.</p>
<p>New basic parts (constructed; awaiting sequencing results):</p>
@@ -222,15 +222,15 @@
<tr><td>BBa_J428346</td><td>Need more markers to transform with different level 1 plasmids</td><td>engineer plasmid for AmpR</td><td>BBa_K4435301</td><td>Design</td></tr>
<tr><td>BBa_J428347</td><td>Need more markers to transform with different level 1 plasmids</td><td>engineer plasmid for AmpR</td><td>BBa_K4435302</td><td>Design</td></tr>
<tr><td>BBa_J428349</td><td>Need more markers to transform with different level 1 plasmids</td><td>engineer plasmid for AmpR</td><td>BBa_K4435303</td><td>Design</td></tr>
- <tr><td>BBa_J428366</td><td>SpecR not tested in Komagataibacter</td><td>engineer plasmid for AmpR</td><td>BBa_K4435304</td><td>Design</td></tr>
- <tr class="emphasis-tr"><td>BBa_J428367</td><td>SpecR not tested in Komagataibacter</td><td>engineer plasmid for AmpR</td><td>BBa_K4435305</td><td>Transformed successfully in Komagataeibacter rhaeticus AF1</td></tr>
- <tr><td>BBa_J428369</td><td>SpecR not tested in Komagataibacter</td><td>engineer plasmid for AmpR</td><td></td><td>planning stage</td></tr>
+ <tr><td>BBa_J428366</td><td>SpecR not tested in <i>Komagataeibacter</i></td><td>engineer plasmid for AmpR</td><td>BBa_K4435304</td><td>Design</td></tr>
+ <tr class="emphasis-tr"><td>BBa_J428367</td><td>SpecR not tested in <i>Komagataeibacter</i></td><td>engineer plasmid for AmpR</td><td>BBa_K4435305</td><td>Transformed successfully in <i>Komagataeibacter rhaeticus</i> AF1</td></tr>
+ <tr><td>BBa_J428369</td><td>SpecR not tested in <i>Komagataeibacter</i></td><td>engineer plasmid for AmpR</td><td></td><td>planning stage</td></tr>
<tr><td>BBa_J428346</td><td>Need more markers to transform with different level 1 plasmids</td><td>engineer plasmid for CmR</td><td>BBa_K4435308</td><td>Design</td></tr>
<tr><td>BBa_J428347</td><td>Need more markers to transform with different level 1 plasmids</td><td>engineer plasmid for CmR</td><td>BBa_K4435309</td><td>Design</td></tr>
<tr><td>BBa_J428349</td><td>Need more markers to transform with different level 1 plasmids</td><td>engineer plasmid for CmR</td><td>BBa_K4435310</td><td>Design</td></tr>
- <tr><td>BBa_J428366</td><td>SpecR not tested in Komagataibacter</td><td>engineer plasmid for CmR</td><td>BBa_K4435311</td><td>Design</td></tr>
- <tr><td>BBa_J428367</td><td>SpecR not tested in Komagataibacter</td><td>engineer plasmid for CmR</td><td>BBa_K4435312</td><td>Design</td></tr>
- <tr><td>BBa_J428369</td><td>SpecR not tested in Komagataibacter</td><td>engineer plasmid for CmR</td><td></td><td>planning stage</td></tr>
+ <tr><td>BBa_J428366</td><td>SpecR not tested in <i>Komagataeibacter</i></td><td>engineer plasmid for CmR</td><td>BBa_K4435311</td><td>Design</td></tr>
+ <tr><td>BBa_J428367</td><td>SpecR not tested in <i>Komagataeibacter</i></td><td>engineer plasmid for CmR</td><td>BBa_K4435312</td><td>Design</td></tr>
+ <tr><td>BBa_J428369</td><td>SpecR not tested in <i>Komagataeibacter</i></td><td>engineer plasmid for CmR</td><td></td><td>planning stage</td></tr>
<tr><td>BBa_J428366</td><td>MCS not compatible</td><td></td><td>BBa_K4435315</td><td>Design</td></tr>
<tr><td>BBa_J428367</td><td>MCS not compatible</td><td></td><td>BBa_K4435316</td><td>Design</td></tr>
<tr><td>BBa_J428369</td><td>MCS not compatible</td><td></td><td>BBa_K4435317</td><td>Design</td></tr>
diff --git a/wiki/pages/results.html b/wiki/pages/results.html
index f12a14d32320e0e8613d0bce13c9a4950eb2b8e8..51dfc0fddf314d92ecea231b34a08d48362486a2 100644
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+++ b/wiki/pages/results.html
@@ -28,7 +28,7 @@
<h2>Construction of broad species spectrum plasmids with compatible selection markers</h2>
(further details in the parts section)
-<p>Komagataeibacter is a non-conventional chassis in which traditional <i>E. coli</i> replication origins are not functional. Hence, plasmid replication requires broad-spectrum origins such as RK2, pBBR1 or RSF1010. Within iGEM´s 2022 distribution kit there were 6 plasmids with such requirements, namely the GoldenGate level 1: BBa_J428346, BBa_J428347, and BBa_J428349 (KanR), and the GoldenGate level 2: BBa_J428366, BBa_J428367, and BBa_J428369 (SpecR). This posed a potential problem to our design strategy as, to the best of our knowledge, there is no report of successful use of the SpecR marker in <i>K. rhaeticus</i>. Thus, we design a strategy to replace the KanR and SpecR marker from level 1 and 2 plasmids (respectively) with both AmpR and CmR. This was accomplished by designing the primers JUMP_mF and JUMP_mR to amplify BBa_J428346, BBa_J428347, BBa_J428349, BBa_J428366, BBa_J428367 and BBa_J428369 excluding the KanR and SpecR selection markers (for details, see parts section). In parallel, we designed primers AmpR_F and AmpR_R to amplify the Ampicillin resistance cassette from BBa_J428385 and primers CmR_F and CmR_R to amplify the chloramphenicol resistance cassette from BBa_J428357. Employing digestion with SapI and ligation with T4 DNA ligase, we succeeded in assembling new level 1 and level 2 Komagateibacter compatible plasmids with different marker options.</p>
+<p><i>Komagataeibacter</i> is a non-conventional chassis in which traditional <i>E. coli</i> replication origins are not functional. Hence, plasmid replication requires broad-spectrum origins such as RK2, pBBR1 or RSF1010. Within iGEM´s 2022 distribution kit there were 6 plasmids with such requirements, namely the GoldenGate level 1: BBa_J428346, BBa_J428347, and BBa_J428349 (KanR), and the GoldenGate level 2: BBa_J428366, BBa_J428367, and BBa_J428369 (SpecR). This posed a potential problem to our design strategy as, to the best of our knowledge, there is no report of successful use of the SpecR marker in <i>K. rhaeticus</i>. Thus, we design a strategy to replace the KanR and SpecR marker from level 1 and 2 plasmids (respectively) with both AmpR and CmR. This was accomplished by designing the primers JUMP_mF and JUMP_mR to amplify BBa_J428346, BBa_J428347, BBa_J428349, BBa_J428366, BBa_J428367 and BBa_J428369 excluding the KanR and SpecR selection markers (for details, see parts section). In parallel, we designed primers AmpR_F and AmpR_R to amplify the Ampicillin resistance cassette from BBa_J428385 and primers CmR_F and CmR_R to amplify the chloramphenicol resistance cassette from BBa_J428357. Employing digestion with SapI and ligation with T4 DNA ligase, we succeeded in assembling new level 1 and level 2 Komagateibacter compatible plasmids with different marker options.</p>
<figure class="figures-left">
<img src="https://static.igem.wiki/teams/4435/wiki/pages/results/results-figura-2.png">
@@ -65,7 +65,7 @@
<figcaption>Figure 3. <i>E. coli</i> transformed with swapped markers plasmids growing in media containing antibiotics.</figcaption>
</figure>
-<p>Our final proof of success is the transformation of Komagataeibacter with part BBa_K4435305, which has the new antibiotic resistance cassette and shows it’s efficiency in <i>Komagataeibacter rhaeticus</i> AF1 (5 and 10 mg/L ampicillin allowed background growth of cells electroporated but without plasmids; 400 mg/L - the recommended ampicillin concentration in the literature - exceeded the tolerable levels for the AF1 strain; 25 mg/L of ampicillin did not allow the growth of cells without plasmids but selected positive transformants). All the new plasmids with swapped markers are await for sequencing.</p>
+<p>Our final proof of success is the transformation of <i>Komagataeibacter</i> with part BBa_K4435305, which has the new antibiotic resistance cassette and shows it’s efficiency in <i>Komagataeibacter rhaeticus</i> AF1 (5 and 10 mg/L ampicillin allowed background growth of cells electroporated but without plasmids; 400 mg/L - the recommended ampicillin concentration in the literature - exceeded the tolerable levels for the AF1 strain; 25 mg/L of ampicillin did not allow the growth of cells without plasmids but selected positive transformants). All the new plasmids with swapped markers are await for sequencing.</p>
<figure class="figures-left">
<img src="https://static.igem.wiki/teams/4435/wiki/pages/results/results-figura-3.png">
@@ -151,7 +151,7 @@
<h3>1. standard media (HS)</h3>
-<p>Komagataeibacter was grown in liquid HS media, which has D-glucose as its sugar source. We asked ourselves if the bacteria use this glucose as a substrate for cellulose production. So, in order to evaluate glucose consumption, we used a quantification technique based on High Performance Liquid Chromatography (HPLC).</p>
+<p><i>Komagataeibacter</i> was grown in liquid HS media, which has D-glucose as its sugar source. We asked ourselves if the bacteria use this glucose as a substrate for cellulose production. So, in order to evaluate glucose consumption, we used a quantification technique based on High Performance Liquid Chromatography (HPLC).</p>
<p>For this analysis, we injected 5 samples collected from different days of bacterial growth, to compare their sugar elution profile to a standard sample of monosaccharides and short-chain carbohydrates. This way it was possible to identify and quantify the sugars present in our sample by integrating their corresponding peaks.</p>
@@ -161,7 +161,7 @@
<figure class="figures-left">
<img src="https://static.igem.wiki/teams/4435/wiki/pages/results/carbohydratequantification.svg">
- <figcaption>Figure 9. Glucose and cellotriose concentrations through the days of Komagataeibacter’s growth in HS liquid media.</figcaption>
+ <figcaption>Figure 9. Glucose and cellotriose concentrations through the days of <i>Komagataeibacter</i>’s growth in HS liquid media.</figcaption>
</figure>
@@ -211,7 +211,7 @@
<li>Fricke, P.M., Klemm, A., Bott, M. et al. On the way toward regulatable expression systems in acetic acid bacteria: target gene expression and use cases. Appl Microbiol Biotechnol 105, 3423–3456 (2021).</li>
<li>Hur DH, Choi WS, Kim TY, Lee SY, Park* JH, Jeong* KJ. Enhanced Production of Bacterial Cellulose in <i>Komagataeibacter xylinus</i> via Tuning of Biosynthesis Genes with Synthetic RBS. J. Microbiol. Biotechnol. 2020;30:1430-1435.</li>
<li>Madhushree Bhattacharya, Melina M. Malinen, Patrick Lauren, Yan-Ru Lou, Saara W. Kuisma, Liisa Kanninen, Martina Lille, Anne Corlu, Christiane GuGuen-Guillouzo, Olli Ikkala, Antti Laukkanen, Arto Urtti, Marjo Yliperttula, Nanofibrillar cellulose hydrogel promotes three-dimensional liver cell culture, Journal of Controlled Release, Volume 164, Issue 3, 2012,Pages 291-298</li>
- <li>Komagataeibacter Tool Kit (KTK): A Modular Cloning System for Multigene Constructs and Programmed Protein Secretion from Cellulose Producing Bacteria Vivianne J. Goosens, Kenneth T. Walker, Silvia M. Aragon, Amritpal Singh, Vivek R. Senthivel, Linda Dekker, Joaquin Caro-Astorga, Marianne L. A. Buat, Wenzhe Song, Koon-Yang Lee, and Tom Ellis ACS Synthetic Biology 2021 10 </li>
+ <li><i>Komagataeibacter</i> Tool Kit (KTK): A Modular Cloning System for Multigene Constructs and Programmed Protein Secretion from Cellulose Producing Bacteria Vivianne J. Goosens, Kenneth T. Walker, Silvia M. Aragon, Amritpal Singh, Vivek R. Senthivel, Linda Dekker, Joaquin Caro-Astorga, Marianne L. A. Buat, Wenzhe Song, Koon-Yang Lee, and Tom Ellis ACS Synthetic Biology 2021 10 </li>
</ul>
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