Difference between revisions of "Team:Newcastle/Notebook/Operon"

 
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                     Naringenin Operon Biosynthesis Notebook
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                     Naringenin Operon
 
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                 <h3 class="subhead">NOTEBOOK</h3>
 
                 <h3 class="subhead">NOTEBOOK</h3>
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                 <h1 class="display-2">Follow the Newcastle iGEM team on their journey</h1>
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                 <h1 class="display-2">Naringenin Operon</h1>
 
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                     <h3 class="h2">Week commencing -06/08/18</h3>
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                     <h3 class="h2">Week commencing - 06/08/18</h3>
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                     <p> The gblocks previously constructed in Benchling based on the parts used by the 2014 TU Darmstadt iGEM team . However we had to manually codon optimised the gene TAL from the operon to lower the G/C content, allowing all 4 gblocks to be ordered from IDT. These gblocks and the primers designed for detection of the assembled operon arrived, therefore lab work could commence. The pSB1C3 backbone for naringenin biosynthesis was amplified using PCR. As the plasmid came with a stock concentration of 25 ng/µl and the PCR reaction requires between 1pg – 1ng the plasmid was diluted by X50. A negative control with 1 µl H2O instead of plasmid DNA was made up. Thermocycling conditions of 71 °C for annealing based on the primers.
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                     <p> A four-gene operon coding for naringenin biosynthesis was designed using Benchling, this was based on the parts used by the 2014 TU Darmstadt iGEM team. For synthesis by IDT, manual codon optimisation was required for the TAL gene to lower the G/C content. Following this, 4 gBlocks were ordered from IDT. These gBlocks and the primers designed for detection of the assembled operon arrived, therefore lab work could commence. The pSB1C3 backbone for naringenin biosynthesis was amplified using PCR. As the plasmid came at a stock concentration of 25 ng/µl and the PCR reaction requires between 1 pg – 1 ng the plasmid was diluted by X50. A negative control with 1 µl H<sub>2</sub>O instead of plasmid DNA was made up. An annealing temperature of 71 °C was used, determined by the NEB TM Calculator. Conditions for PCR are shown in the table below. Agarose gel electrophoresis was used for detection of the 2 kb amplified band, this was then purified using a QIAquick PCR Purification Kit, however, it resulted in a very low concentration of DNA around 3.58 µg/ml. Therefore, it was planned to conduct this PCR again to increase the concentration.
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An overnight culture of DH5α cells was inoculated to make more competent cells for subsequent transformations. These cells were grown in a 37 °C shaking incubator until the OD<sub)600</sub> reaches 0.2 – 0.3, followed by additions of 75 mM MgCl<sub)2</sub>, 75 mM CaCl<sub)2</sub> and 15 % glycerol to induce competency.
  
 
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                     <h3 class="h2">Day 2 - 08/06/18</h3>
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                     <h3 class="h2">Week commencing - 13/08/18</h3>
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                     <p>The day began with a talk from Jon Marles-Wright about the importance of human practices and the impacts of
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                     <p>A positive control for Gibson assembly was undertaken by assembling 2 overlapping dsDNA fragments and the backbone pUC19 control DNA provided by NEB, using the NEBuilder HiFi DNA Assembly Cloning Kit. This was used to ensure that Gibson assembly using the master mix worked, and that the cells had been made competent. This positive control reaction was then transformed into the competent cells made earlier in the week using NEBuilder HiFi DNA Assembly Transformation Protocol. These cells underwent heat shock before being spread onto LB plates containing ampicillin as the positive control plasmid carried resistance to ampicillin. All 3 of the positive control plates had growth of DH5α cells, the negative control plate had no growth of DH5α cells, as they contained no plasmid and were not ampicillin resistant. This showed that the Gibson master mix was effective and the competent cells had been successfully made competent.
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                    our projects in the real world. This was followed by Dana Ofiteru who taught us about mathematical modeling of
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                    biological systems. In the afternoon, plant biologist Max Kapralov discussed using synthetic biology to improve
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                    the efficiency of photosynthesis. Finally, Rachel Armstrong (professor of experimental architecture) discussed
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                    the potential of microorganism in waste management systems and energy production in the built environment. Rachel
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                    also outlined political and economic issues surrounding synthetic biology.
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                     <h3 class="h2">Day 3 - 11/06/18</h3>
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                     <h3 class="h2">Week commencing - 20/08/18</h3>
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                     <p>We started the day with a talk from Angel Goni Moreno who discussed the principles of synthetic biology and provided
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                     <p>gBlocks and the primers for the detection of the gBlocks were resuspended following instructions by IDT. Gibson assembly of the gBlocks and the pSB1C3 backbone was conducted using the volumes calculated through NEB: the vector should be 0.05 pmol and each insert between 0.1 – 0.2 pmol so that the total does not exceed 0.5 pmol. Competent cells were then transformed using the reaction mix and plated onto LB agar containing chloramphenicol instead of previously used ampicillin, as pSB1C3 has chloramphenicol resistance. Following overnight incubation, no colonies were observed on either the test plates of the negative control plates.
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                    guidance on the implementation of logic in Biology. The Masters students who are working on the Interlab study explained
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                    their role in the project and we brainstormed ideas. Following this, the Synthetic Biology Masters students discussed
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                    their individual projects involving modelling lab work. Each of the team gave a presentation on a previous competition
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                    entry that has won an award.
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                     <h3 class="h2">Day 4- 12/06/18</h3>
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                     <h3 class="h2">Week commencing- 27/08/18</h3>
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                     <p>We started the day with a talk from Alice banks and Colette Whitfield. Followed by brainstorming with Tom, refining
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                     <p>A second attempt at Gibson assembly was conducted and the transformation protocol was performed the same as previously. No colony growth resulted from this. It was considered that the backbone concentration was too low, which could be limiting transformation. More of the backbone was amplified and purified to a stock concentration of 26.2 µg/ ml.
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                    the original ideas with some extra. Then the marine biologist/Engineered talked to us about boat fouling. Finally we had a
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                    talk from Vas Andriotis who focused on plant biology (corn yield). Finishing with brainstorming.
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                     <h3 class="h2">Day 5- 13/06/18</h3>
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                     <h3 class="h2">Week commencing- 3/09/18</h3>
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                     <p>The day began with a talk from Martyn Dade-Robertson who discussed the use of biotechnology in architecture and his research
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                     <p>A third attempt of Gibson assembly was done using the increased backbone concentration resulting in a total reaction volume of 36.3 µl. This was then transformed into DH5α cells resulting in 1 colony on each plate where  200 µl of transformation mix had been plated. However, these colonies only showed that the pSB1C3 backbone had been take up by the cells due to the observed chloramphenicol resistance. In order to test for the correct naringenin operon assembly, colony PCR was undertaken. This utilised primers 4CLF and CHSR to amplify from the start and end of the operon with 66 °C annealing temperature. This reaction should amplify the full operon if it has been inserted into the backbone correctly, this would result in a PCR product of ~5 kb. A 3 minute extension time was required for amplification the large fragment. Following analysis of PCR products by agarose gel electrophoresis, no 5 kb band could be seen on the gel. It was considered that no band was observed due to inherent difficulties with amplifying large PCR products. As an alternative means of analysis, overnight cultures of selected colonies were prepared to undergo plasmid extraction by miniprep. Plasmids were extracted from overnight cultures of two colonies using Qiagen QIAprep Plasmid Miniprep Kit. Extracted plasmids were analysed by agarose gel electrophoresis where a band of ~7 kb was expected if the Gibson assembly had been successful.
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                    into pressure sensing E.coli. In the afternoon Harold Freemann led a discussion around the use of mathematical modelling and its
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                      uses in synthetic biology. Finally Alice Banks and Colette Whitfield covered current topics in synthetic biology and the  
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                      equipment available to us in the laboratory.
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                     <h3 class="h2">Day 6- 14/06/18</h3>
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                     <h3 class="h2">Week commencing- 10/09/18</h3>
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                     <p>This was our first day in which we were given freedom to explore our ideas and gain a necessary background surrounding
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                     <p>Following agarose gel electrophoresis, the only bands present for multiple samples of the two colonies were at 2 kb showing only the uptake of the plasmid from which the pSB1C3 had been amplified. Next, Gibson assembly was conducted again, however, instead of having an 18 µl reaction, the mixture was spun down in a SpeedVac 120 for 5 minutes to increase the DNA concentration. This was performed so that the Gibson assembly reaction volume could be reduced to the recommended 20 µl. 5 µl of the Gibson assembly was used to transform DH5α cells, this was increased from the 2 µl used previously. However, following overnight incubation, there was no colony growth on any of the plates. Primers were designed for the amplification of the gBlocks and ordered from IDT so that the parts could be amplified for use in additional Gibson assemblies.
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                    the topics in order to refine into 6 ideas that will be pitched to research supervisors and the guest speakers who have
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                    given presentations since the project began.
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                     <h3 class="h2">Day 7- 15/06/18</h3>
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                     <h3 class="h2">Week commencing- 17/09/18</h3>
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                     <p>We continued to explore our ideas for the majority of the day, narrowing our proposals down from ten ideas to six.  
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                     <p>The new primers for gBlock amplification arrived and were used to amplify the gBlocks by PCR. A gradient PCR was attempted to try a range of annealing temperatures and a 1 minute extension time was used. Analysis by agarose gel electrophoresis showed that gBlocks 3 and 4 and the pSB1C3 backbone amplified well resulting in bright bands at the expected sizes. However, the amplification of gBlock 1 resulted in two bands on the agarose gel and this raised concerns over contamination. gBlock 2 had not successfully amplified, and no band was observed on the agarose gel. To troubleshoot this, a gradient PCR was attempted across a range of different annealing temperatures, this resulted in 8 reactions (Table 4). Reaction 3 of the gBlock 2 amplification showed the clearest band, therefore this was purified and the DNA concentration quantified which determined a concentration of 51.8 µg/ml. Gel extraction was performed on the other bands amplified from gBlock 2, as there was uncertainty as to which band was correct. Two bands were obtained for gBlock 1, so both of these were gel extracted to recover the PCR product. The stock concentrations of all purified fragments are shown in Table 5. A series of Gibson assembly reactions were performed using different combinations of the amplified products. Reaction mixes were used to transform commercial TOP10 chemically competent cells (Thermo Fisher Scientific) and between 30-60 colonies grew on each plate following overnight incubation on selective medium.
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                    We worked more as individuals, researching specific aspects of the proposals.
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                     <h3 class="h2">Day 8- 18/06/18</h3>
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                     <h3 class="h2">Week commencing- 24/09/18</h3>
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                     <p>Today was more orientated around the ethical implications Synthetic Biology has. We started with a discussion with Dr.  
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                     <p>Overnight cultures for 3 colonies from each of the 4 plates were grown and plasmids were extracted from the resultant cultures. Plasmid extractions were analysed by agarose gel electrophoresis (Figure 4). Only the backbone had been taken up by the cells. An additional 10 more colonies were analysed in the same way. An additional Gibson assembly was also attempted and two transformations of commercial TOP10 <i>E. coli</i> were carried out, one using 2 µl of reaction mix and one with 5 µl, each reaction used 50 µl of cells. Unfortunately, these attempts did not yield any colonies. The plasmids extracted from the additional 10 colonies were ran on agarose gel, however only 2 kb bands were observed. An additional 10 colonies were analysed in the same way. Agarose gel electrophoresis showed a promising band at 7 kb for the miniprep of colony 19 (Figure 5), which was the correct size of the plasmid and the operon. In order to reduce the observed level of background resulting from the plasmid backbone, the PCR amplification of the pSB1C3 backbone was treated with DpnI (NEB) and purified to a stock concentration of 9.84 µg/ml. A subsequent attempt at Gibson assembly was then attempted using 6.53 µl of the DpnI treated backbone. To check the results of the miniprep of colony 19, PCR was performed using the primers 4CLF and CHSR in an attempt to amplify the 5 kb operon. Additional PCR was performed using primers for the detection of each gBlock (Table 6).
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                    Ilke Turkendag, a lecturer in Law Innovation & Society. We discussed our six proposals and outlines possible ethical/legal
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                    issues which could arise. This was followed by an interesting talk from Simon Woods and Ken Taylor, they spoke about potential
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                    stakeholders and how we could go about talking/communicating with them. They also took each of our ideas and looked at the  
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                    potential impacts our projects could have on the wider public. We then finished the day preparing for our six pitches on Tuesday.
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The OpenTrons OT-2 robot, which the Newcastle iGEM team won, was also delivered today<sup>1</sup>.
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                     <h3 class="h2">Day 9- 19/06/18</h3>
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                     <h3 class="h2">Week commencing- 1/10/18</h3>
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                     <p>We pitched the six ideas that had been selected to the advisers and masters students. After receiving feedback and suggestions
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                     <p>All PCR products were analysed by agarose gel electrophoresis, however, no bands were observed on the gel. Despite this, colony 19 was sent for sequencing as the plasmid was of the expected size (Eurofins Genomics). This plasmid was also used to transform <i>E. coli</i> BL21(DE3) cells for potential future expression analysis. In concordance with this, the Gibson assembly with the DpnI treated backbone was used to transform TOP10 and DH5α commercial <i>E. coli</i> cells. This resulted in lots of colonies on the DH5α cell plates and less than 5 on each TOP10 plate. Colonies 21-25 from the Gibson assemblies using variants of gel extracted and amplified gBlocks were transferred to overnight cultures, alongside colonies 27-34 transformations with the DpnI treated Gibson assembly mix. Minipreps were performed on overnight cultures of colonies 21-34, and one band was observed at 7 kb for colony 23 when analysed by agarose gel electrophoresis. This was subsequently sent for sequencing. Plasmids extracted from a further 10 colonies were analysed, however, all were found to contain the template plasmid from the pSB1C3 amplification. Sequencing data for the two plasmids sequenced did not provide sufficient coverage to confirm the correct assembly of the naringenin operon into the pSB1C3 backbone.
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                    from the advisory team we narrowed it down to two ideas to be taken forward. Further we constructed a template for both idea
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                    presentations and allocated team members to each idea. Umar is now our least favorite team member :)
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                    <h3 class="h2">Day 10/11- 21/06/18</h3>
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                    <p>Following on from the 2 ideas that we chose to take forward, we spent these next 2 days researching further into these proposals.
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                    During our research, we took a holistic approach when evaluating our ideas and attempted to account for topics such as human practices,
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                    ethical implications and possible art and design ideas. We incorporated this into our 2 ideas which we then pitched to the supervisory
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                    team and Master's students. We also managed to successfully setup the OT-2 robot. The smiles speak for themselves...
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                    <h3 class="h2">Day 12- 22/06/18</h3>
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                    <p>This day was used to carefully consider both ideas that were pitched the previous day before voting on one to take forward.
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                    We started by summarizing each idea in 140 characters which highlighted some discrepancies in individual team members understanding
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                    of the ideas. After these discrepancies were rectified, the team spent some time considering each idea before voting on one to take
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                    up as our project. In the end, it was decided that the project involving engineering microbes to colonize plant roots and attract
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                    nitrogen-fixing bacteria was the best to take forward. We spent some time thinking about the modeling/human practices/lab work that
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                    may be involved in this project.
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                    <h3 class="h2">Day 13- 25/06/18</h3>
+
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                    <p>On our first official day of project-specific work, various tasks were allocated to team members for them to research.
+
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                    Some background reading was conducted on the topic, the team name was confirmed and some ideas for the team logo were suggested.
+
−
                    Also, some ideas for a hydroponics system were discussed. With a rare bit of sunshine, we decided to work outdoors.
+
−
                    </p>
+
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<img src="https://static.igem.org/mediawiki/2018/c/cb/T--Newcastle--June25thPic.jpeg">
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            </div>
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                    <h3 class="h2">Week commencing: 25/06/18</h3>
+
−
                    <p>On the Monday, members of the team were provided an overview of the plant growth facilities at Newcastle University by
+
−
                    Vas Andriotis who has shown great interest in our project since its pitch and has given many resources to enhance
+
−
                    and develop our ideas. The remainder of the group began on the research for the several experiments that will be ran in
+
−
                    parallel over the Summer. <br>
+
−
 
+
−
Tuesday was dedicated to further fleshing out our research by identifying species which would be suitable for our iGEM project.
+
−
Some members moved in an artistic direction and produced a handful of potential logos before the entire team voted on our two
+
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favorites *put logos here*. Once these were decided, merchandising began development. <br>
+
−
 
+
−
Development on our hydroponics system moved into full swing on the Wednesday, with our 2 engineers beginning work on the general
+
−
structure of the system which has been influenced by conversations with plant biologists within the university; working once again
+
−
with Vas in order to understand his needs from the system. <br>
+
−
 
+
−
Thursday presented our team with the incredible opportunity to speak with Steven Burgess who is currently performing plant research
+
−
at the University of Illinois in order to discuss our project and identify potential for sharing our work among the wider
+
−
scientific community. <br>
+
−
 
+
−
Members of the Biology and Engineering teams went to meet with Dr Montero-Calasanz; a Microbiologist whom specialises in plant-microbe
+
−
interactions. This gave a valuable opportunity to identify potential protocols which would relate to our chassis organism. The week was
+
−
rounded off by talks from Alice Banks and Collette Whitfield concerning sponsorship and outreach.
+
−
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                    <img src="https://static.igem.org/mediawiki/2018/thumb/7/7e/T--Newcastle--Logos2.png/267px-T--Newcastle--Logos2.png">
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                    <h3 class="h2">Week commencing: 02/07/18</h3>
+
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                    <p>The team focused on preparation for the wetware projects by ordering the <i>Pseudomonas</i> chassis, in addition to various free-living
+
−
nitrogen-fixing bacteria. A variety of flavinoid chemoattractants were also ordered in order to reduce the wait time before lab work begins.
+
−
The human practices and outreach team continued getting their work by in touch with potential sponsors. Some of the team members with prior
+
−
experience in programming began working with the OT-2 to design a heat-shock protocol for <i>E. coli</i> using the python-based language within the
+
−
OpenTrons API.
+
−
The whole team received inductions for the Cat2 lab so that we can begin the wetware side of the project and follow all the safety protocols
+
−
and rules and regulations set out by the University for safe lab use.
+
−
After receiving the lab inductions, we prepared the lab equipment for testing <i>Arabidopsis</i> germination so that we can begin testing the following week.
+
−
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                    <img src="https://static.igem.org/mediawiki/2018/8/8b/T--Newcastle--July12thPic.jpeg">
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                    <h3 class="h2">Week commencing: 09/07/18</h3>
+
−
                    <p>The ball started to roll this week, we managed to get access to the labs and plant our first set of seeds in agar as well as
+
−
                    preparing competent <i> E. coli</i> cells. We began the process of styling our wiki using bootstraps and designing the poster
+
−
                    for the European Meetup. Towards the end of the week we attended a modeling workshop, ran by Synthetic Biology Masters students,
+
−
                    this gave us an insight into modelling and tips we can apply to our modeling work. The team was also lucky enough to recieve goodiebags
+
−
                    of Eppendorf
+
−
We had previously decided that the naringenin flavonoid would most likely be the most successful, therefore looked into utilising parts from the naringenin operon previously used by TU Darmstadt 2014 iGEM team. We also Skyped Daniel Sachs, a member of the TU Darmstadt iGEM team to hear his advice on what worked well with the operon and with which promoters. Finally, we codon optimised one of the gblocks for the operon (TAL) and ordered all the segments with their corresponding RBS’s from IDT.<sup>2</sup>.
+
−
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+
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                    <video controls><source src="https://static.igem.org/mediawiki/2018/4/45/T--Newcastle--OTUnbox.mp4" type="video/mp4">
+
−
  </video>
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                    <h3 class="h2">Week commencing 16/07/2018</h3>
+
−
                    <p>This week was full of action. The architects in the team 3D printed a cold deck for the OpenTrons OT-2 and laser cut accompanying trays to hold test tubes. Now that we have the cold deck we can manufacture trays to hold lots of different apparatus increasing the number and complexity of experiments we can carry out on the OpenTrons. In addition, we had a meeting with Martyn Dade-Robertson from the School of Architecture, Planning & Landscape; he spoke to us about the importance of finding a context in which to situate our human practices, once we have this it will provide the design parameters necessary to progress. Finally, stencils were made for the 'Alternative Roots' logo enabling us to tag any hardware we build. <br>
+
−
 
+
−
Some of the team also travelled to Munich this week for the European iGEM meetup. Those who attended throughly enjoyed
+
−
liasing with other iGEM teams as well as the event organisers and speakers. The team's poster was on display at the
+
−
meetup which gathered some interest around our project as well as many questions and suggestions for improvement which
+
−
have been duly noted by the team for further consideration. Potential collaborations were also sought after in Munich. <br>
+
−
 
+
−
Lab work for the chemotaxis assays commenced with trial runs using the DH5α E. coli control in order to test
+
−
the protocol. It was noted from these attempt that the concentration of naringenin was too high as the plates
+
−
did not show signs of growth for 2 days; after which, signs of growth were minimal.
+
−
The concentration was diluted to 100µM and 50µM for future trials to see if this would overcome the issue. We also Designed forward and reverse primers for each of the four segments, for detection that the operon had been transformed.
+
−
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+
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                    <img src="https://static.igem.org/mediawiki/2018/thumb/c/ce/T--Newcastle--ColdDeckTray.png/180px-T--Newcastle--ColdDeckTray.png.jpeg">
+
−
                    <img src="https://static.igem.org/mediawiki/2018/thumb/f/f6/T--Newcastle--ColdDeck.png/180px-T--Newcastle--ColdDeck.png.jpeg">
+
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                    <img src="https://static.igem.org/mediawiki/2018/thumb/1/1a/T--Newcastle--Munich.png/320px-T--Newcastle--Munich.png" width=365px>
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                    <h3 class="h2">Week commencing 23/07/2018</h3>
+
−
                    <p>The long-awaited <i>Pseudomonas fluroescens</i> arrived this week,having come all the way from Germany,
+
−
meaning our iGEM team could start preparation for the transformation process. The team worked with
+
−
Dr Montero-Calasanz to make initial agar and liquid cultures in both LB and TS mediums and now
+
−
have enough bacteria to start the initial transformation experiments.
+
−
<br>
+
−
This week the some of the parts for the hardware arrived meaning we could get underway with the
+
−
construction of the hydroponics. First the basic circuit for the LED lights was created, the code
+
−
we used to control these lights was an adaptation of a freely available library (FastLED) which can
+
−
be found online. We made minor adjustments to suit our requirements, for example the
+
−
light intensity was increased. Our micro-controller (Arduino), was then loaded with
+
−
this edited code. This device will be responsible for monitoring and maintaining our entire
+
−
system. We faced issues with timing the lights for a 16 hour day and 8 hour night cycle.
+
−
We decided to use a ‘count’ within a loop to control when the LED’s need to be turned off.
+
−
This meant we had to calibrate for the time a single loop took and multiply that up for
+
−
16 hours. We are currently waiting for some parts to arrive so that we can begin construction of an
+
−
automated switching circuit.
+
−
<br>
+
−
 
+
−
 
+
−
The week was rounded off with a presentation to the stakeholders in the project, such as the PI's
+
−
and advisers, to provide an update on how the project has progressed thus far. The presentation
+
−
also offered a valuable opportunity to highlight areas of the project that needed to be strengthened
+
−
and was a good opportunity to develop our presentation skills prior to the Jamboree in Boston.
+
−
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+
−
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+
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                    <h3 class="h2">Week commencing 30/07/2018</h3>
+
−
                    <p>This week we managed to get off campus and talk to potential stakeholders. We were lucky enough to arrange an interview with GrowModule 365. As a company, they produce shipping containers that contain hydroponics systems. There concept adopts a new approach to farming, allowing crop production in unconventional places. The visit allowed us to see how traditional farming is being challenged and gain an insight into hydroponics systems. We gained lots of useful information from the meeting; we learned which wavelengths are most effective for increased yields, the alternative growing mediums available, the markets perception to innovative farming and which control parameters are most important. Paul Brown (Director) gave us lots of useful contacts within the industry and seemed very passionate about our project, however he did mention some areas of the market may be sceptical when it comes to eating produce that has been grown using genetically engineered microbes.
+
−
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+
−
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+
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                    <h3 class="h2">Week commencing 06/08/2018</h3>
+
−
                    <p>This week we started to work on a video for the opening of the presentation in Boston. We wanted to continue with the theme portrayed in a previous 'teaser' we produced. The hydroponics switching circuit was finalised by the engineers, this controlled the day and night cycle to simulate specific environments for the seeds. The Hydroponics system aesthetics were also worked on, we applied spray paint and stencils to make it more visually appealing. At the end of the week MMatt Burridge (One of our masters students) conducted a workshop to teach people how to write and run protocols on the OOpentrons OT-2 robot.
+
−
<br>
+
−
In the lab we: amplified the backbone of pSBS1C3 through thermocycling using primers for the 2kb sequence required for assembly and this allowed the plasmid to be linearized and prepare for later insertion of the gblocks for the naringenin operon,
+
−
ran this PCR product using agarose gel electrophoresis to check for separation of the backbone required from the rest of the plasmid, purified the PCR product and quantified the DNA concentration thus making the backbone ready for Gibson Assembly.
+
−
Towards the end of the week amplified the positive control for Gibson assembly practice using the NEBuilder HiFi DNA Assembly Cloning Kit.
+
−
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+
−
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                    <img src="https://static.igem.org/mediawiki/2018/thumb/0/05/T--Newcastle--OT2Talk.jpeg/300px-T--Newcastle--OT2Talk.jpeg" width=3000>
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                    <h3 class="h2">Week commencing 13/08/2018</h3>
+
−
                    <p>  We were in the final stages of building the hydroponics system this week, we have determined optimal wavelengths to run the system at. Chris 3D printed a series of trays for seeds to fit in. We were also lucky enough to have a meeting with Chris Tapsell From KWS (the 4th largest seed compay in the world). We outlined our project and discussed the issues surrounding GM crops and how the industry and wider public respond to GM use. Chris seemed to be passionate about the project and we intend to stay in contact with each other.
+
−
<br>
+
−
In the lab we made competent DH5α E. coli cells for Gibson transformation through the use of repeated centrifugations and the reagents MgCl2, CaCl2 and Glycerol.
+
−
We also transformed these chemically competent cells with the positive control from Gibson assembly and found that the Gibson Assembly method was successful, as the positive control grew on ampicillin plates due to the DH5α cells taking up the positive control overlapping dsDNA fragments containing ampicillin resistance. No growth was observed on the negative control plates with un- transformed DH5α cells, as they are not naturally resistant to ampicillin.
+
−
 
+
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                    <img src="https://static.igem.org/mediawiki/2018/thumb/e/e5/T--Newcastle--Hydroponicssystem.jpeg/800px-T--Newcastle--Hydroponicssystem.jpeg" width=3000>
+
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+
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                    <h3 class="h2">Week commencing 20/08/2018</h3>
+
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                    <p>The week started with a visit to Scotland to meet the Edinburgh iGEM team. The visit was extremely useful as it allowed us to share our progress with Edinburgh and vice versa and we also gave each other feedback on our respective projects. Additionally, we discussed the potential for collaborations, ate lots of pizza and even got to see some of the shows in the Edinburgh Fringe festival. We thoroughly enjoyed our time in Edinburgh and meeting the Edinburgh team, who were very hospitable and we hope to maintain a close relationship with Edinburgh iGEM moving forward.
+
−
<br>
+
−
Two members of the team, Will and Umar, also travelled to London to meet with Richard Ballard, co-founder of Growing Underground. We were shown around their tunnels of hydroponics which grow approximately 33 metres below the busy streets of Clapham. Growing Underground sustainably grow fresh micro greens and salad leaves, which are unaffected by weather and seasonal changes, before providing these to wholesalers and local restaurants such as M&S and Ocado. The meeting was very productive, we discussed how our project may be beneficial to the company and, also, how the company can help our project develop. We discussed the project at length and considered how the project might be refined to better meet the needs and concerns of a company such as Growing Underground. We hope to continue working with Richard and the Growing Underground team as we continue to develop the project.
+
−
<br>
+
−
This week in the lab, we resuspended the gblocks and primers from IDT. We also calculated the volumes of pSB1C3 and gblock inserts required for specific concentrations in the Gibson assembly and conducted a Gibson Assembly using the NEBuilder HiFi DNA Assembly Reaction Protocol. We then transformed the Gibson Assembly using the NEBuilder HiFi DNA Assembly Protocol, and then spread the reaction mixture onto chloramphenicol selection plates for overnight incubation. There was no colony growth on any of the plates therefore transformation was unsuccessful.
+
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+
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                    <h3 class="h2">Week commencing 27/08/2018</h3>
+
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                    <p>On the chemotaxis side of things, we spent the week producing kill curves for each of our bacteria as to determine whether our concentration of naringenin was having an adverse effect on the growth of our bacteria. This was as we knew naringenin possessed antimicrobial properties thus it was important to work out if this was the reason that we found chemorepulsion for E. coli instead of no effect. During this week we also secured a sponsorship from ibidi who we are happy to say provided the team with their specialised μ-slideIII 3-in-1 chemotaxis slide which allowed us to visualise cell movement in response to naringenin via timelapse videos. It was also during this week that we reinvented our chemotaxis assay to be more in line with the work of Reyes-Darias et al. (2014) by reducing our agar percentage even further and utilising large, square plates in order to run multiple replicates and test distances on a single plate.
+
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<br>
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We also conducted a second attempt of Gibson with re – calculated values for the inserts. This was transformed as before and there was no growth on the plates. As this was thought to be due to the backbone concentration being far too low after purification, another PCR to linearize the backbone was conducted. This was ran on a gel and quantified to have a much higher concentration.
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                    <h3 class="h2">Week commencing 03/09/2018</h3>
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                    <p>This week we carried out our reinvented chemotaxis assays that involved using minimal agar on square plates, and setting up a concentration gradient of naringenin, and obtained results which showed positive chemotaxis of <i>Azospirillum brasilens</i> towards 100uM narinegenin at distances between 0.5cm and 2.5cm! The most successful result was when the bacteria was at a distance of 1.5cm, so we will carry out repeats of this distance.
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A third Gibson attempt with the new backbone and the gblocks was done. Following transformation this reaction yielded 2 colonies showing that the plasmid with chloramphenicol resistance had been taken up. In order to see whether the rest of the operon had been integrated into the plasmid, colony PCR and plasmid miniprep was conducted on both colonies. However only the plasmid had been taken up without the operon.
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                    <h3 class="h2">Week commencing 10/09/2018</h3>
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                    <p>This week we were able to successfully transform <i>Pseudomonas fluorescens</i> with a gentamicin resistance gene by electroporation.
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We also tested methods of sterilising plant tissue, such as using different concentrations of ethanol and bleach. Seeds and seedlings were inoculated with wild type <i>P. fluorescens</i>.
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Performed square plate assays with <i>Herbaspirillum seropedicae</i> and <i>Azorhizobium caulinodans</i>, but results were inconclusive as colonies grew too large and overlapped. Repeats will be performed with bacteria inoculated at greater distances from each other.
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As wiki freeze draws closer, we are trying to develop more pages of our wiki and keep on top of it to hopefully lighten the load later when the deadline becomes imminent.
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                    <h5>Acknowledgements</h5>
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                    <p>1. Credit: <a href="https://twitter.com/alice_banks/status/1008710700485799941" target="_blank">Dr. Alice Banks</a>
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                      2. Credit: <a href="https://twitter.com/JoshuaLoh6/status/1009456142320328705" target="_blank">Joshua Loh</a>
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                <h1 class="display-2">References & Attributions</h1>
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<p class="about-para"><font size="2"><strong>Attributions: Heather Bottomley and Patricya Ubysz
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Latest revision as of 17:29, 17 October 2018

Alternative Roots/Notebook

Alternative Roots

Naringenin Operon

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NOTEBOOK

Naringenin Operon

Week commencing - 06/08/18

A four-gene operon coding for naringenin biosynthesis was designed using Benchling, this was based on the parts used by the 2014 TU Darmstadt iGEM team. For synthesis by IDT, manual codon optimisation was required for the TAL gene to lower the G/C content. Following this, 4 gBlocks were ordered from IDT. These gBlocks and the primers designed for detection of the assembled operon arrived, therefore lab work could commence. The pSB1C3 backbone for naringenin biosynthesis was amplified using PCR. As the plasmid came at a stock concentration of 25 ng/µl and the PCR reaction requires between 1 pg – 1 ng the plasmid was diluted by X50. A negative control with 1 µl H2O instead of plasmid DNA was made up. An annealing temperature of 71 °C was used, determined by the NEB TM Calculator. Conditions for PCR are shown in the table below. Agarose gel electrophoresis was used for detection of the 2 kb amplified band, this was then purified using a QIAquick PCR Purification Kit, however, it resulted in a very low concentration of DNA around 3.58 µg/ml. Therefore, it was planned to conduct this PCR again to increase the concentration. An overnight culture of DH5α cells was inoculated to make more competent cells for subsequent transformations. These cells were grown in a 37 °C shaking incubator until the OD reaches 0.2 – 0.3, followed by additions of 75 mM MgCl, 75 mM CaCl and 15 % glycerol to induce competency.

Week commencing - 13/08/18

A positive control for Gibson assembly was undertaken by assembling 2 overlapping dsDNA fragments and the backbone pUC19 control DNA provided by NEB, using the NEBuilder HiFi DNA Assembly Cloning Kit. This was used to ensure that Gibson assembly using the master mix worked, and that the cells had been made competent. This positive control reaction was then transformed into the competent cells made earlier in the week using NEBuilder HiFi DNA Assembly Transformation Protocol. These cells underwent heat shock before being spread onto LB plates containing ampicillin as the positive control plasmid carried resistance to ampicillin. All 3 of the positive control plates had growth of DH5α cells, the negative control plate had no growth of DH5α cells, as they contained no plasmid and were not ampicillin resistant. This showed that the Gibson master mix was effective and the competent cells had been successfully made competent.

Week commencing - 20/08/18

gBlocks and the primers for the detection of the gBlocks were resuspended following instructions by IDT. Gibson assembly of the gBlocks and the pSB1C3 backbone was conducted using the volumes calculated through NEB: the vector should be 0.05 pmol and each insert between 0.1 – 0.2 pmol so that the total does not exceed 0.5 pmol. Competent cells were then transformed using the reaction mix and plated onto LB agar containing chloramphenicol instead of previously used ampicillin, as pSB1C3 has chloramphenicol resistance. Following overnight incubation, no colonies were observed on either the test plates of the negative control plates.

Week commencing- 27/08/18

A second attempt at Gibson assembly was conducted and the transformation protocol was performed the same as previously. No colony growth resulted from this. It was considered that the backbone concentration was too low, which could be limiting transformation. More of the backbone was amplified and purified to a stock concentration of 26.2 µg/ ml.

Week commencing- 3/09/18

A third attempt of Gibson assembly was done using the increased backbone concentration resulting in a total reaction volume of 36.3 µl. This was then transformed into DH5α cells resulting in 1 colony on each plate where 200 µl of transformation mix had been plated. However, these colonies only showed that the pSB1C3 backbone had been take up by the cells due to the observed chloramphenicol resistance. In order to test for the correct naringenin operon assembly, colony PCR was undertaken. This utilised primers 4CLF and CHSR to amplify from the start and end of the operon with 66 °C annealing temperature. This reaction should amplify the full operon if it has been inserted into the backbone correctly, this would result in a PCR product of ~5 kb. A 3 minute extension time was required for amplification the large fragment. Following analysis of PCR products by agarose gel electrophoresis, no 5 kb band could be seen on the gel. It was considered that no band was observed due to inherent difficulties with amplifying large PCR products. As an alternative means of analysis, overnight cultures of selected colonies were prepared to undergo plasmid extraction by miniprep. Plasmids were extracted from overnight cultures of two colonies using Qiagen QIAprep Plasmid Miniprep Kit. Extracted plasmids were analysed by agarose gel electrophoresis where a band of ~7 kb was expected if the Gibson assembly had been successful.

Week commencing- 10/09/18

Following agarose gel electrophoresis, the only bands present for multiple samples of the two colonies were at 2 kb showing only the uptake of the plasmid from which the pSB1C3 had been amplified. Next, Gibson assembly was conducted again, however, instead of having an 18 µl reaction, the mixture was spun down in a SpeedVac 120 for 5 minutes to increase the DNA concentration. This was performed so that the Gibson assembly reaction volume could be reduced to the recommended 20 µl. 5 µl of the Gibson assembly was used to transform DH5α cells, this was increased from the 2 µl used previously. However, following overnight incubation, there was no colony growth on any of the plates. Primers were designed for the amplification of the gBlocks and ordered from IDT so that the parts could be amplified for use in additional Gibson assemblies.

Week commencing- 17/09/18

The new primers for gBlock amplification arrived and were used to amplify the gBlocks by PCR. A gradient PCR was attempted to try a range of annealing temperatures and a 1 minute extension time was used. Analysis by agarose gel electrophoresis showed that gBlocks 3 and 4 and the pSB1C3 backbone amplified well resulting in bright bands at the expected sizes. However, the amplification of gBlock 1 resulted in two bands on the agarose gel and this raised concerns over contamination. gBlock 2 had not successfully amplified, and no band was observed on the agarose gel. To troubleshoot this, a gradient PCR was attempted across a range of different annealing temperatures, this resulted in 8 reactions (Table 4). Reaction 3 of the gBlock 2 amplification showed the clearest band, therefore this was purified and the DNA concentration quantified which determined a concentration of 51.8 µg/ml. Gel extraction was performed on the other bands amplified from gBlock 2, as there was uncertainty as to which band was correct. Two bands were obtained for gBlock 1, so both of these were gel extracted to recover the PCR product. The stock concentrations of all purified fragments are shown in Table 5. A series of Gibson assembly reactions were performed using different combinations of the amplified products. Reaction mixes were used to transform commercial TOP10 chemically competent cells (Thermo Fisher Scientific) and between 30-60 colonies grew on each plate following overnight incubation on selective medium.

Week commencing- 24/09/18

Overnight cultures for 3 colonies from each of the 4 plates were grown and plasmids were extracted from the resultant cultures. Plasmid extractions were analysed by agarose gel electrophoresis (Figure 4). Only the backbone had been taken up by the cells. An additional 10 more colonies were analysed in the same way. An additional Gibson assembly was also attempted and two transformations of commercial TOP10 E. coli were carried out, one using 2 µl of reaction mix and one with 5 µl, each reaction used 50 µl of cells. Unfortunately, these attempts did not yield any colonies. The plasmids extracted from the additional 10 colonies were ran on agarose gel, however only 2 kb bands were observed. An additional 10 colonies were analysed in the same way. Agarose gel electrophoresis showed a promising band at 7 kb for the miniprep of colony 19 (Figure 5), which was the correct size of the plasmid and the operon. In order to reduce the observed level of background resulting from the plasmid backbone, the PCR amplification of the pSB1C3 backbone was treated with DpnI (NEB) and purified to a stock concentration of 9.84 µg/ml. A subsequent attempt at Gibson assembly was then attempted using 6.53 µl of the DpnI treated backbone. To check the results of the miniprep of colony 19, PCR was performed using the primers 4CLF and CHSR in an attempt to amplify the 5 kb operon. Additional PCR was performed using primers for the detection of each gBlock (Table 6).

Week commencing- 1/10/18

All PCR products were analysed by agarose gel electrophoresis, however, no bands were observed on the gel. Despite this, colony 19 was sent for sequencing as the plasmid was of the expected size (Eurofins Genomics). This plasmid was also used to transform E. coli BL21(DE3) cells for potential future expression analysis. In concordance with this, the Gibson assembly with the DpnI treated backbone was used to transform TOP10 and DH5α commercial E. coli cells. This resulted in lots of colonies on the DH5α cell plates and less than 5 on each TOP10 plate. Colonies 21-25 from the Gibson assemblies using variants of gel extracted and amplified gBlocks were transferred to overnight cultures, alongside colonies 27-34 transformations with the DpnI treated Gibson assembly mix. Minipreps were performed on overnight cultures of colonies 21-34, and one band was observed at 7 kb for colony 23 when analysed by agarose gel electrophoresis. This was subsequently sent for sequencing. Plasmids extracted from a further 10 colonies were analysed, however, all were found to contain the template plasmid from the pSB1C3 amplification. Sequencing data for the two plasmids sequenced did not provide sufficient coverage to confirm the correct assembly of the naringenin operon into the pSB1C3 backbone.





References & Attributions

Attributions: Heather Bottomley and Patricya Ubysz