Template:Team:SydneyUni Australia/Calendar/Events List

description: 'Rob<br>Robbie introduces iGEM to the Manager of Inspiring Australia in NSW, facilitator for National Science Week. Advice '},

description: 'Rob, Andrew, Viv, Shuravi, Cyril, Desmond, Hugh, Evelyn<br>First team meeting at Hermann’s. We talked about some crazy outreach ideas like gfp graffiti and organizing a symposium. The seed that became Strange Nature was planted. A lot of support for DCA degradation and Botany Bay as our lab project. '},

description: 'Rob, Andrew<br>Attend the CLC Town Hall Meeting held by Orica at Botany Bay. We introduce iGEM and our project and look for possible collaborators. ' },

description: 'Nick, Rob, Andrew, Viv, Shuravi, Cyril, Desmond, Hugh, Evelyn<br>Learned that we won’t be able to use Mox from Xanthobacter autotrophicus as planned due to the reliance on co-factor PQQ. Began considering a monooxygenase pathway for degradation.  ' },

description: 'Nick, Rob, Andrew, Viv, Shuravi, Cyril, Desmond, Hugh, Evelyn<br>Met with Yagiz and Rob from the MQ University iGEM team for a general introduction to iGEM and some helpful advice.'},

description: 'Nick, Rob, Andrew, Viv, Shuravi, Cyril, Desmond, Hugh, Evelyn<br>Tutorial on primer design for designing primers for the project'},

description: 'Coleman, Viv, Desmond, Rob and Hugh<br>We made some solutions of LB, LB agar and TE. We staked out our claim in the lab. Yay!'},

description: 'Coleman, Andrew, Cyril<br>Continued the ToMO plasmid prep. Reached the step where DNA is precipitated in ethanol and acetate overnight'},

description: 'Coleman, Viv, Rob<br>Finished the ToMO plasmid prep. Digested pBBR1-MCS2. Used the nanodrop. Did a digest + gel of our ToMO, undigested and Xbal1-digested pBBR along with a marker'},

description: 'Coleman, Cyril, Shuravi, Rob<br>We transformed E. coli Epi300 with two plasmids, pBBR and pBS(ToMO). As both have Km resistance, only those cells that were successfully transformed with the plasmid would grown when plated onto LB+Km media. They were incubated overnight'},

description: 'Nick, Cyril, Andrew, Rob<br>We discussed Gibson Assembly and gBlocks for the first time, due to concerns about having enough time.'},

description: 'Coleman, Andrew, Desmond<br>The two E. Coli treatments were growing at different rates, E. Coli (pBS-ToMO) wasn’t growing well at all. We transferred E. Coli (pBBR) to broth and later washed and transferred to two phosphate buffer treatments, one with DCA, one without. We transferred E. Coli (pBS-ToMO) to broth and incubated it overnight. DCA concentration was 1mM. '},

description: 'Coleman, Rob, Hugh<br>After 24 hours in DCA, we put E. Coli (pBBR) in the cool room for a Cl assay next week. We washed and transferred E. Coli (pBS-ToMO) from broth to two phosphate buffer treatments, one with DCA, one without. We also ran a quick PCR testing our new ToMO primers on pBS-ToMO, but we didn’t digest pBS-ToMO before PCR. !'},

description: 'Coleman, Rob<br>We repeated the ToMO trial however made some changes to the protocol.'},

description: 'Rob, Andrew, Viv, Shuravi, Desmond, Cyril<br>Lasertag and bowling with the Macquarie University iGEM team.'},

description: 'Coleman, Cyril, Desmond, Shuravi<br>Performed chloride assay on ToMO, pBBR, TOM/+-DCA samples. ToMO came 1st place, with reasonable amounts of DCA metabolised.'},

description: 'Elissa, Cyril, Desmond, Rob<br>We tried to PCR the origin of replication from pBBR1-MCS2 using primer iGEM 5 and 6. We digested the plasmid with EcoR1 ran a diagnostic PCR but didn’t see anything. We re-digested the plasmid with a higher concentration of DNA, and did a gradient PCR, and didn’t see the desired PCR product. We’re looking into mispriming elsewhere on pBBR.'},

description: 'Nick, Rob, Andrew, Viv, Shuravi, Desmond, Evelyn<br>Met to dicuss Gibson Assembly and decided to pursue this method of assembly. Delegated some tasks for finding sequences, optimisation and ordering of gBlocks from IDT using the special iGEM discount.'},

description: 'Shuravi, Rob<br>Prepared for arrival of gBlocks by making up reagents for plasmid prep, pouring chloramphenicol. We also tested whether the prepared PstQ cells were competent and uncontaminated'},

description: 'Rob, Hugh<br>- The following morning (13th), nothing had grown. Our positive control showed that the transformation was successful, negative control that our technique was sterile.<br><br>- Ran a gel of pSB1C3 (the plasmid that the gBlocks are being assembled into), to confirm that we were sent a clean sample by the iGEM HQ.<br><br>- The Gibson Assembly was run again with only a positive control (a pUC19 plasmid with 5 gBlocks, provided by IDT), transformed and incubated overnight. The following morning  (14th) a few, small colonies were growing on ampicillin plates, indicating the pUC19 positive control has assembled correctly.' },

description: 'Rob, Hugh, Shuravi, Desmond<br>A few different things were tried including:<br><br>- Transforming into a different E. Coli host (TOP10), since perhaps our home-made cells weren’t up to IDT’s standards.<br><br>- Transforming into the same host (EPI300) but letting the cells recover and incubate at room temperature. Perhaps our pathway assembled correctly but overexpression of foreign enzymes impeded cells at maximum growth rate. Results were the same as the original Gibson Assembly transformation.'},

description: 'Rob, Vivian<br>Set-up 87 clones to incubate in chloroacetate overnight to detect Cl- release, indicating the presence of dhlB in pSB1C3.'},

{ title: 'Chloride Assay',

description: 'Rob, Andrew, Desmond<br>[Cl-] assay of the 87 clones<br><br>Chose 10 of the clones based on the results (5 representing each pathway) and set-up broths for a plasmid prep and a second [Cl-] assay (taking cells from plates instead of broth).'},

description: 'Viv<br>Treated plasmids with RNAse, repeated final steps of plasmid prep.'},

description: 'Andrew, Nick<br>- Plated out cells carrying pUC19-dhlB for positive control in [Cl-] assay.<br><br>- Inoculated 4ml broths for [Cl-] assay'},

description: 'Rob<br>Another transformation was performed using the second set of Gibson assembly products into E.coli TOP 10 and E.coli Epi400.  The transformed cells were plated onto the appropriate antibiotic media.'},

description: 'Rob<br>A diagnostic digest was performed on our old plasmid preps. p-p was digested with BamH1, MluI, SmaI, SphI and Sal1. P-a digested with MluI, SmaI, SphI and rfp had no restriction enzyme added'},

description: 'Andrew<br>A plasmid prep was performed on pSB-RFP, taken from transformed E.coli TOP10 cells. Stopped at precipitation stage.<br><br>We received our new set of primers! We used these to PCR screen all our selected Gibson assembly plasmids and GA restriction products and our positive GA control (puc19-dhlA-dhlB)<br><br>To confirm that our PCR screening had worked we ran a gel of all our PCR products'},

description: 'Rob, Andrew, James<br>Following on from yesterdays digested plasmid prep a gel was run to at different concentrations.<br><br>We found, from the gel of our digest, that the plasmids were not concentrated enough.<br><br>Another plasmid prep, using some different clones was done. This time however, we incubated cells overnight to increase plasmid yield.<br><br>We digested and ran a gel of our plasmid for confirmation.<br><br><div style=\'color:red;font-weight:bold;\'>The gel turned out perfect!</div>'},

description: 'Rob, Andrew<br>Construction of inducible promoter system in pSB1C3. Ran out of luck with lacI and turned to arabinose and tetracycline systems. PCR of parts from the Distribution Kit and verification of length and purity on gels.<br><br>Started playing around with the ingredients of chloroacetate-phenol red screening plates. We did a quick titration to find an optimal pH close to the colour-change from phenol red.'},

description: 'Everyone<br>Ohh no!! Wikifreeze!'},

        end: new Date(2013, 9, 2),

description: 'Everyone<br>Presentations all day!'},

description: 'Everyone<br>Won the Best Human Practices and Best Experimental Measurement Approach!'},

description: 'Rob, Andrew<br>Convincing our supervisor to purchase the new gBlocks and primers we need.'},

description: 'Hugh, Shuravi<br>Transformation of more clones with pSB1C3-dhlB ligation mixture. PCR Screen for correct orientation of dhlB from non-directional ligation. '},

description: 'Rob, Desmond<br>Plating pSB1C3-Ptet-dhlB clones on pH-chloroacetate plates to find clones degrading chloroacetate. Set-up an overnight incubation of resting cells to confirm chloroacetate degradation.'},

{ title: 'Further confirmation',

description: 'James<br>Chloride assay to confirm chloroacetate degradation. Woo! We’ve got dhlB isolated and it works!'},

description: 'Rob, Andrew, Shuravi<br>More screening. Plasmid prep of promising clones.'},

description: 'Rob, Andrew, Hugh<br>Digest confirmation of plasmid with a RE specific to each gBlock. Confirmed length and concentration of all new parts. Digestion, gel purification, ligation and transformation of new parts so that they can be expressed.'},

description: 'Andrew<br>Transformation plates hadn’t grown sufficiently for screening.'},

description: 'Rob, Andrew<br>Another LB-acetaldehyde growth experiment in triplicate with a promising tetR-aldA clone. Incubated tetR-p450 clones with DCA in triplicate to confirm result from the 24th October, after 6 hours incubation, GC was not promising. Set-up an ethene-epoxide assay to confirm p450 expression.'},

description: 'Viv, Rob, James, Hugh<br>Sat together and updated the wiki for changes to project made since Hong Kong'}