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Template:Team:SydneyUni Australia/Calendar/Events List
From 2013.igem.org
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title: 'First Meeting', | title: 'First Meeting', | ||
start: new Date(2012, 12, 31), | start: new Date(2012, 12, 31), | ||
| - | description: ' | + | description: 'Coleman, Rob<br>Robbie introduces iGEM to potential supervisors around campus. Coleman pricks his ears. ' |
}, | }, | ||
{ | { | ||
title: 'Advisor Meeting', | title: 'Advisor Meeting', | ||
start: new Date(2013, 2, 14), | start: new Date(2013, 2, 14), | ||
| - | description: ' | + | description: 'Rob<br>Robbie introduces iGEM to the Manager of Inspiring Australia in NSW, facilitator for National Science Week. Advice ' |
}, | }, | ||
{ | { | ||
title: 'First Team Meeting', | title: 'First Team Meeting', | ||
start: new Date(2013, 2, 18), | start: new Date(2013, 2, 18), | ||
| - | description: ' | + | 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. ' |
}, | }, | ||
{ | { | ||
title: 'CLC meeting', | title: 'CLC meeting', | ||
start: new Date(2013, 2, 26), | start: new Date(2013, 2, 26), | ||
| - | description: ' | + | 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. ' |
}, | }, | ||
{ | { | ||
title: 'Project planning', | title: 'Project planning', | ||
start: new Date(2013, 3, 9), | start: new Date(2013, 3, 9), | ||
| - | description: ' | + | 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. ' |
}, | }, | ||
{ | { | ||
title: 'Meeting Yagiz', | title: 'Meeting Yagiz', | ||
start: new Date(2013, 4, 3), | start: new Date(2013, 4, 3), | ||
| - | description: ' | + | 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. ' |
}, | }, | ||
{ | { | ||
title: 'Tutorial on primer design. ', | title: 'Tutorial on primer design. ', | ||
start: new Date(2013, 4, 14), | start: new Date(2013, 4, 14), | ||
| - | description: ' | + | description: 'Nick, Rob, Andrew, Viv, Shuravi, Cyril, Desmond, Hugh, Evelyn<br>Tutorial on primer design for designing primers for the project' |
}, | }, | ||
// -----------------Week 1 ---------------- | // -----------------Week 1 ---------------- | ||
| Line 61: | Line 61: | ||
title: 'Lab Setup', | title: 'Lab Setup', | ||
start: new Date(2013, 4, 17), | start: new Date(2013, 4, 17), | ||
| - | description: ' | + | 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!' |
}, | }, | ||
{ | { | ||
title: 'ToMO plasmid Extraction', | title: 'ToMO plasmid Extraction', | ||
start: new Date(2013, 4, 22), | start: new Date(2013, 4, 22), | ||
| - | description: ' | + | description: 'Coleman, Andrew, Desmond and Shuravi<br>We started the plasmid extraction of ToMO from the E. coli.' |
}, | }, | ||
{ | { | ||
title: 'ToMO plasmid Extraction', | title: 'ToMO plasmid Extraction', | ||
start: new Date(2013, 4, 23), | start: new Date(2013, 4, 23), | ||
| - | description: ' | + | description: 'Coleman, Andrew, Cyril<br>Continued the ToMO plasmid prep. Reached the step where DNA is precipitated in ethanol and acetate overnight' |
}, | }, | ||
{ | { | ||
title: 'ToMO plasmid Extraction', | title: 'ToMO plasmid Extraction', | ||
start: new Date(2013, 4, 24), | start: new Date(2013, 4, 24), | ||
| - | description: ' | + | 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' |
}, | }, | ||
// -----------------Week 2 ---------------- | // -----------------Week 2 ---------------- | ||
| Line 82: | Line 82: | ||
title: 'Transformation and Selection', | title: 'Transformation and Selection', | ||
start: new Date(2013, 4, 29), | start: new Date(2013, 4, 29), | ||
| - | description: ' | + | 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' |
}, | }, | ||
{ | { | ||
title: 'Intro to Gibson', | title: 'Intro to Gibson', | ||
start: new Date(2013, 4, 29), | start: new Date(2013, 4, 29), | ||
| - | description: ' | + | description: 'Nick, Cyril, Andrew, Rob<br>We discussed Gibson Assembly and gBlocks for the first time, due to concerns about having enough time. ' |
}, | }, | ||
{ | { | ||
title: 'Growing up cells', | title: 'Growing up cells', | ||
start: new Date(2013, 4, 30), | start: new Date(2013, 4, 30), | ||
| - | description: ' | + | 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. ' |
}, | }, | ||
{ | { | ||
title: 'Chloride Assay', | title: 'Chloride Assay', | ||
start: new Date(2013, 4, 31), | start: new Date(2013, 4, 31), | ||
| - | description: ' | + | 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. !' |
}, | }, | ||
// -----------------Week 3 ---------------- | // -----------------Week 3 ---------------- | ||
| Line 103: | Line 103: | ||
title: 'Meeting with Yagiz', | title: 'Meeting with Yagiz', | ||
start: new Date(2013, 5, 2), | start: new Date(2013, 5, 2), | ||
| - | description: ' | + | description: 'Rob<br>We met with Yagiz at Macquarie University to talk about Strange Nature and possible collaboration with our iGEM teams.' |
}, | }, | ||
{ | { | ||
title: 'PCR analysis and GC analysis of DCA metabolism', | title: 'PCR analysis and GC analysis of DCA metabolism', | ||
start: new Date(2013, 5, 3), | start: new Date(2013, 5, 3), | ||
| - | description: ' | + | description: 'Coleman, Hugh, Rob<br>We ran a gel of last Friday’s PCR. The PCR was of pBS(ToMO) extracted from Wood’s E. Coli TG1, with primers iGEM 1 & 2. The banding on the gel was obscure, potentially due to too much template and could not be used.<br><br>We used the Gas Chromatographer (GC) to look at our two DCA treatments of E. Coli (transformed with either pBBR or pBS(ToMO)). Both had pretty much the same reading but it is uncertain yet whether this is because pBS(ToMO) was lost or altered during the extended incubation from Week 2, or whether ToMO was unable to degrade DCA. We also set up standardised solutions of NaCl in KP buffer for a future Cl assay.' |
}, | }, | ||
// -----------------Week 4 ---------------- | // -----------------Week 4 ---------------- | ||
| Line 116: | Line 116: | ||
start: new Date(2013, 5, 22), | start: new Date(2013, 5, 22), | ||
end: new Date(2013, 5, 27), | end: new Date(2013, 5, 27), | ||
| - | description: ' | + | description: 'Coleman, Rob<br>We repeated the ToMO trial however made some changes to the protocol. ' |
}, | }, | ||
// -----------------Week 5 ---------------- | // -----------------Week 5 ---------------- | ||
| Line 122: | Line 122: | ||
title: 'Meeting Mac Uni Team', | title: 'Meeting Mac Uni Team', | ||
start: new Date(2013, 6, 1), | start: new Date(2013, 6, 1), | ||
| - | description: ' | + | description: 'Rob, Andrew, Viv, Shuravi, Desmond, Cyril<br>Lasertag and bowling with the Macquarie University iGEM team. ' |
}, | }, | ||
{ | { | ||
title: 'ToMO Chloride Assay', | title: 'ToMO Chloride Assay', | ||
start: new Date(2013, 6, 4), | start: new Date(2013, 6, 4), | ||
| - | description: ' | + | 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. ' |
}, | }, | ||
// -----------------Week 6 ---------------- | // -----------------Week 6 ---------------- | ||
| Line 134: | Line 134: | ||
start: new Date(2013, 6, 11), | start: new Date(2013, 6, 11), | ||
end: new Date(2013, 6, 12), | end: new Date(2013, 6, 12), | ||
| - | description: ' | + | 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. ' |
}, | }, | ||
{ | { | ||
title: 'Gibson Planning', | title: 'Gibson Planning', | ||
start: new Date(2013, 6, 12), | start: new Date(2013, 6, 12), | ||
| - | description: ' | + | 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. ' |
}, | }, | ||
// -----------------Week 7 ---------------- | // -----------------Week 7 ---------------- | ||
| Line 146: | Line 146: | ||
start: new Date(2013, 6, 23), | start: new Date(2013, 6, 23), | ||
end: new Date(2013, 6, 24), | end: new Date(2013, 6, 24), | ||
| - | description: ' | + | description: 'Desmond, Rob<br>We made competent PstQ for electroporation (Pseudomonas stutzeri, strain Q, the ones that are naturally competent for transformation, but we will also be testing our genes as plasmids).' |
}, | }, | ||
{ | { | ||
title: 'General Lab Stuff', | title: 'General Lab Stuff', | ||
start: new Date(2013, 6, 26), | start: new Date(2013, 6, 26), | ||
| - | description: ' | + | 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 ' |
}, | }, | ||
// -----------------Week 8 ---------------- | // -----------------Week 8 ---------------- | ||
| Line 157: | Line 157: | ||
title: 'Gibson Assembly', | title: 'Gibson Assembly', | ||
start: new Date(2013, 7, 12), | start: new Date(2013, 7, 12), | ||
| - | description: ' | + | description: 'Andrew, Rob<br>gBlocks arrived at the end of last week. We did a Gibson Assembly reaction, and transformed the product into E. Coli (EPI300). These were incubated overnight.' |
}, | }, | ||
{ | { | ||
title: 'Checking Gibson', | title: 'Checking Gibson', | ||
start: new Date(2013, 7, 13), | start: new Date(2013, 7, 13), | ||
| - | description: ' | + | 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.' |
}, | }, | ||
{ | { | ||
| Line 168: | Line 168: | ||
start: new Date(2013, 7, 14), | start: new Date(2013, 7, 14), | ||
end: new Date(2013, 7, 15), | end: new Date(2013, 7, 15), | ||
| - | description: ' | + | 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.' |
}, | }, | ||
// -----------------Week 9 ---------------- | // -----------------Week 9 ---------------- | ||
| Line 175: | Line 175: | ||
start: new Date(2013, 7, 16), | start: new Date(2013, 7, 16), | ||
end: new Date(2013, 7, 18), | end: new Date(2013, 7, 18), | ||
| - | description: ' | + | description: 'Rob, Nick, Desmond, Andrew<br>- Checked plates and learned that transforming our Gibson Assembly product into TOP10 (rather than EPI300) worked better than before.<br><br>- This was confirmed on Saturday morning (17th) after re-transforming the same reaction with the last of our Gibson Assembly product, in order to obtain more colonies for screening (for finding which of the colonies contains the correctly assembled pathway).<br><br>- A master plate containing all colonies was patched for phenotypic screening the following week.' |
}, | }, | ||
{ | { | ||
title: 'Chloride Assay', | title: 'Chloride Assay', | ||
start: new Date(2013, 7, 19), | start: new Date(2013, 7, 19), | ||
| - | description: ' | + | 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', | title: 'Chloride Assay', | ||
start: new Date(2013, 7, 20), | start: new Date(2013, 7, 20), | ||
| - | description: ' | + | 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). ' |
}, | }, | ||
{ | { | ||
title: 'Chloride Assay, Plasmid Prep', | title: 'Chloride Assay, Plasmid Prep', | ||
start: new Date(2013, 7, 21), | start: new Date(2013, 7, 21), | ||
| - | description: ' | + | description: 'Rob, Shuravi, Hugh<br>[Cl-] assayed the 10 clones. Plasmid prepped the 10 clones.' |
}, | }, | ||
{ | { | ||
title: 'Gel of Plasmid Prep', | title: 'Gel of Plasmid Prep', | ||
start: new Date(2013, 7, 22), | start: new Date(2013, 7, 22), | ||
| - | description: ' | + | description: 'Rob, Nick<br>Ran a gel of yesterday’s plasmid prep and found it was too diluted and full of RNA.' |
}, | }, | ||
{ | { | ||
title: 'Plasmid Prep Take 2', | title: 'Plasmid Prep Take 2', | ||
start: new Date(2013, 7, 23), | start: new Date(2013, 7, 23), | ||
| - | description: ' | + | description: 'Viv<br>Treated plasmids with RNAse, repeated final steps of plasmid prep. ' |
}, | }, | ||
// -----------------Week 10 ---------------- | // -----------------Week 10 ---------------- | ||
| Line 206: | Line 206: | ||
start: new Date(2013, 7, 24), | start: new Date(2013, 7, 24), | ||
end: new Date(2013, 7, 25), | end: new Date(2013, 7, 25), | ||
| - | description: ' | + | description: 'Andrew, Nick<br>- Plated out cells carrying pUC19-dhlB for positive control in [Cl-] assay.<br><br>- Inoculated 4ml broths for [Cl-] assay' |
}, | }, | ||
{ | { | ||
title: 'Chloride Assay', | title: 'Chloride Assay', | ||
start: new Date(2013, 7, 26), | start: new Date(2013, 7, 26), | ||
| - | description: ' | + | description: 'Desmond<br>- Transferred overnight cultures (4mL) to fresh 50 mL LB and grown to an OD of ~1.0. Cells were harvested, cleaned and resuspended in 2mM chloroacetate for overnight incubation for chloride assay the next day.' |
}, | }, | ||
{ | { | ||
title: 'Chloride Assay Stuff', | title: 'Chloride Assay Stuff', | ||
start: new Date(2013, 7, 27), | start: new Date(2013, 7, 27), | ||
| - | description: ' | + | description: 'Rob<br>Remaining E.coli was spread plated onto Mackonkey agar for future negative Cl- assays.<br><br>Solutions for plasmid preps and chloride assays were made. We suspect the ambiguous plasmid prep and chloride assays results are due to poor stocks.' |
}, | }, | ||
{ | { | ||
title: 'Chloride Assay', | title: 'Chloride Assay', | ||
start: new Date(2013, 7, 28), | start: new Date(2013, 7, 28), | ||
| - | description: ' | + | description: 'Rob<br>Inoculated LB-Cm with our clones [2,10,38, 42,54,64, rfp(63)], LB-Am (positive control) and LB (negative control) for plasmid prep and chloride assay. No growth was observed in positive and negative control so no culture could be transferred for chloride assay.' |
}, | }, | ||
{ | { | ||
title: 'Chloride Assay Stuff', | title: 'Chloride Assay Stuff', | ||
start: new Date(2013, 7, 29), | start: new Date(2013, 7, 29), | ||
| - | description: ' | + | description: 'Rob, Shuravi, Desmond<br>Clones grown in LB-antibiotic broth overnight were washed and added to 2mM DCA reagents and another set in 2mM chloroacetate reagent. Standards using NaCl solution was made simultaneously to ensure accuracy of the assay.<br><br>Plasmid preps were done using clones 2, 10, 38, 54, 42, 64 and rfp 63.<br><br>The samples were patch plated onto new LB-Cm plates.' |
}, | }, | ||
// -----------------Week 11 ---------------- | // -----------------Week 11 ---------------- | ||
| Line 232: | Line 232: | ||
title: 'Diagnostic Gel', | title: 'Diagnostic Gel', | ||
start: new Date(2013, 7, 31), | start: new Date(2013, 7, 31), | ||
| - | description: ' | + | description: 'Rob<br>A diagnostic digest was set up for clones 42, 54 and 64 using EcoRV and 2, 38, rfp using EcrIVHF. Banding patterns on our gel was difficult to see but was better visualised under UV.' |
}, | }, | ||
{ | { | ||
title: 'PCR Work', | title: 'PCR Work', | ||
start: new Date(2013, 8, 2), | start: new Date(2013, 8, 2), | ||
| - | description: ' | + | description: 'Rob, James<br>Our new primers arrived. Today we PCR screened for dhlB and found the reverse primer corresponded to the synthetic dhlB sequence. Our PCR products were then loaded onto gel. The results were a little disappointing as there was no banding on our first gel and the ladder was difficult to see. Primer dimers were observed on our gel.<br><br>We also inoculated another 20 clones for screening from our initial Gibson Assembly transformation of p-p [contains p450] and p-a [contains adh] (These are our two different pathways).<br><br>We prepared some competent EPI400 cells. These are to be used later for the transformation of our GA products.' |
}, | }, | ||
{ | { | ||
title: 'Cyril\'s Birthday', | title: 'Cyril\'s Birthday', | ||
start: new Date(2013, 8, 2), | start: new Date(2013, 8, 2), | ||
| - | description: ' | + | description: 'Cyril<br><b>Happy Birthday :) </b>' |
}, | }, | ||
{ | { | ||
title: 'Cell Transformation, Gibson Assembly', | title: 'Cell Transformation, Gibson Assembly', | ||
start: new Date(2013, 8, 3), | start: new Date(2013, 8, 3), | ||
| - | description: ' | + | description: 'Andrew, Rob<br>We transformed our Epi400 cells with our Gibson assembly products. Results did not look very promising. The Epi400 cells looked as they did when we transformed Epi300. The control worked and few cells grew with the positive Gibson assembly. There was more growth of cells transformed with p-p and p-a.<br><br>Another Gibson Assembly was set up for transformation. We found iGEMblock1 did not contain enough DNA leaving less than needed for the p-a sample.<br><br>We PCR screened for dhlB in colonies 100-139 however did not have a positive control (did not work yesterday). A gel was run for the dhlB PCR products.' |
}, | }, | ||
{ | { | ||
title: 'Cell Transformation', | title: 'Cell Transformation', | ||
start: new Date(2013, 8, 4), | start: new Date(2013, 8, 4), | ||
| - | description: ' | + | 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. ' |
}, | }, | ||
{ | { | ||
title: 'PCR and Diagnostic Gel', | title: 'PCR and Diagnostic Gel', | ||
start: new Date(2013, 8, 5), | start: new Date(2013, 8, 5), | ||
| - | description: ' | + | description: 'Andrew<br>We did a PCR of dhlB from the GA reaction products using three samples (p-p, p-a and a negative control containing no DNA)<br><br>All GA enzymes were heat-killed by placing using thermocycler at 95 degrees.<br><br>We ran a gel of dhlB PCR of Gibson assembly products. The resulting bands were clear and as expected.' |
}, | }, | ||
{ | { | ||
title: 'Diagnostic Gel', | title: 'Diagnostic Gel', | ||
start: new Date(2013, 8, 6), | start: new Date(2013, 8, 6), | ||
| - | description: ' | + | 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' |
}, | }, | ||
// -----------------Week 12 ---------------- | // -----------------Week 12 ---------------- | ||
| Line 268: | Line 268: | ||
title: 'Digests', | title: 'Digests', | ||
start: new Date(2013, 8, 9), | start: new Date(2013, 8, 9), | ||
| - | description: ' | + | description: 'Andrew, Desmond, Rob<br>Two digests were set up in parallel (p-p and p-a) using SspI and later purified using Quiaquick columns.' |
}, | }, | ||
{ | { | ||
title: 'Plasmid Prep, PCR Screen', | title: 'Plasmid Prep, PCR Screen', | ||
start: new Date(2013, 8, 10), | start: new Date(2013, 8, 10), | ||
| - | description: ' | + | 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' |
}, | }, | ||
{ | { | ||
title: 'Plasmid Prep, Gradient PCR', | title: 'Plasmid Prep, Gradient PCR', | ||
start: new Date(2013, 8, 11), | start: new Date(2013, 8, 11), | ||
| - | description: ' | + | description: 'Vivian, James, Desmond, Andrew<br>A gradient PCR was done on dhlB and dhlA from our positive GA samples.<br><br>Gels were run for each of our PCR products from the previous day.<br><br>The plasmid prep from earlier in the week was completed. Andrew later digested the Gibson assembly product using sspI. Desmond set up a digest for Rfp using ECRo1 and ran a gel of the digests.<br><br>Another PCR was done using the Gibson assembly digested fragments.' |
}, | }, | ||
{ | { | ||
title: 'Ligation and Transformation', | title: 'Ligation and Transformation', | ||
start: new Date(2013, 8, 12), | start: new Date(2013, 8, 12), | ||
| - | description: ' | + | description: 'Rob, Andrew, Vivian<br>Today a digestion of pSB-rfp was made using the enzymes, xbaI and PstI for ligation. Our PCR fragments (dhlA and dhlB) were also digested using the same restriction enzymes.<br><br>Following purification of our PCR DNA we ligated our inserts (dhlA, dhlB or dhlA-dhlB) into our plasmid vector (pSB). We split the reaction volume to two and use one later by storing in the cool room.<br><br>The ligated products were transformed into E.coli Top10 and EpI300 and plated on to LB-Cm.' |
}, | }, | ||
{ | { | ||
title: 'PCR Screen', | title: 'PCR Screen', | ||
start: new Date(2013, 8, 13), | start: new Date(2013, 8, 13), | ||
| - | description: ' | + | description: 'Rob<br>We PCR screened for the correct dhlA, dhlB, dhlA-dhlB inserts by creating patch plates of white colonies on LB-Cm. Each clone was then mixed with EB and boiled to release DNA from cells. The cell suspension of each clone were used to PCR the DNA. Unfortunately some resulted in no PCR product.' |
}, | }, | ||
{ | { | ||
title: 'Plasmid Prep, More Screening', | title: 'Plasmid Prep, More Screening', | ||
start: new Date(2013, 8, 14), | start: new Date(2013, 8, 14), | ||
| - | description: ' | + | description: 'Rob, Andrew<br>Based on yesterday screens we selected clones for plasmid prepping. Plasmids were digested using EcoRV to check length and concentration of DNA.<br><br>To find successfully ligated dhlB we did more PCR screening. This time however we used EPI300 cells instead of TOP10. Screening was also done for dhlB-dhlA.' |
}, | }, | ||
// -----------------Week 13 ---------------- | // -----------------Week 13 ---------------- | ||
| Line 299: | Line 299: | ||
title: 'Gel and Plasmid Prep (Again)', | title: 'Gel and Plasmid Prep (Again)', | ||
start: new Date(2013, 8, 15), | start: new Date(2013, 8, 15), | ||
| - | description: ' | + | 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>' |
}, | }, | ||
{ | { | ||
title: 'Sending off Parts', | title: 'Sending off Parts', | ||
start: new Date(2013, 8, 16), | start: new Date(2013, 8, 16), | ||
| - | description: ' | + | description: 'Viv, James, Andrew, Rob<br>We sent off our parts for dhlA (BBa_K1115004), dhlB (BBa_K1115005) and dhlB-dhlA (BBa_K1115006), based on the results from PCR screening of cells and digests of plasmid preps.<br><br>However, we did a PCR screen using the plasmids as templates and discovered one of our parts is a reverse insert! This is dhlB (BBa_K1115005).' |
}, | }, | ||
{ | { | ||
title: 'Sending off Parts, Characterisation', | title: 'Sending off Parts, Characterisation', | ||
start: new Date(2013, 8, 17), | start: new Date(2013, 8, 17), | ||
| - | description: ' | + | description: 'Andrew, Rob<br>Re-submitted parts we’re pretty sure are correct. Started setting up cells and reagents for construction of a constitutive and inducible promoter system to characterise our parts in pSB1C#3 before the wiki-freeze.<br><br>PCR of parts directly from Distribution Kit for subsequent use in directional cloning.' |
}, | }, | ||
{ | { | ||
title: 'Transformation and Promoter Hunting', | title: 'Transformation and Promoter Hunting', | ||
start: new Date(2013, 8, 18), | start: new Date(2013, 8, 18), | ||
| - | description: ' | + | description: 'Rob, Shuravi, Viv, Andrew<br>Construction of Pcat-dhlB-dhlA in pSB1C3. Rob wrote out all the protocol and ran a digest, Viv ligated and transformed the construct, Shuravi spread-plated colonies.<br><br>Andrew went through a lot of troubleshooting trying to PCR parts or plasmid prep parts from the Distribution Kit for a lacI inducible promoter.' |
}, | }, | ||
{ | { | ||
title: 'Screening Plates', | title: 'Screening Plates', | ||
start: new Date(2013, 8, 19), | start: new Date(2013, 8, 19), | ||
| - | description: ' | + | description: 'Rob, Andrew<br>Figured out we could make screening plates to help us find transformants that are expressing our construct. We added chloroacetate and phenol red to LB-agar before plating and looked for colour change as the bacteria degraded chloroacetate and released Cl- ions.' |
}, | }, | ||
{ | { | ||
title: 'Promoter and Plate Construction', | title: 'Promoter and Plate Construction', | ||
start: new Date(2013, 8, 20), | start: new Date(2013, 8, 20), | ||
| - | description: ' | + | 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. ' |
}, | }, | ||
{ | { | ||
| Line 330: | Line 330: | ||
start: new Date(2013, 8, 21), | start: new Date(2013, 8, 21), | ||
end: new Date(2013, 8, 22), | end: new Date(2013, 8, 22), | ||
| - | description: ' | + | description: 'Rob, Andrew, James, Viv<br>Sequential digestion and ligation of PCR products for an inducible promoter (Ptet and TetR, Pbad and AraC, parts from Distribution Kit).<br><br>Found that LB-agar-chloramphenicol plates with 10mM chloroacetate, 18mg/L phenol red, pH 6.8, worked best and allowed us to pick a few clones constitutively expressing dhlB for further characterisation by chloride assay.' |
}, | }, | ||
{ | { | ||
title: 'Chloride Assay, Inducibility Screen', | title: 'Chloride Assay, Inducibility Screen', | ||
start: new Date(2013, 8, 23), | start: new Date(2013, 8, 23), | ||
| - | description: ' | + | description: 'Rob, Andrew, Viv<br>Set-up chloride assay for a few promising clones constitutively expressing dhlB.<br><br>Set-up plates using the same screening system to find clones with inducible expression of dhlB.' |
}, | }, | ||
{ | { | ||
title: 'Chloride Assay', | title: 'Chloride Assay', | ||
start: new Date(2013, 8, 24), | start: new Date(2013, 8, 24), | ||
| - | description: ' | + | description: 'Rob, Andrew<br>Chloride assay showed degradation of chloroacetate and DCA using the parts we submitted to the iGEM HQ.' |
}, | }, | ||
{ | { | ||
title: 'Triplicate Chloride Assay', | title: 'Triplicate Chloride Assay', | ||
start: new Date(2013, 8, 25), | start: new Date(2013, 8, 25), | ||
| - | description: ' | + | description: 'Rob, Andrew, James<br>Set-up another chloride assay in triplicate for neat characterisation of our parts, in parallel with promising clones with inducible-promoter systems.<br><br>James ran an SDS-page gel for further evidence that our parts are being expressed.' |
}, | }, | ||
{ | { | ||
title: 'Chloride Assay, Promoter Characterisation', | title: 'Chloride Assay, Promoter Characterisation', | ||
start: new Date(2013, 8, 26), | start: new Date(2013, 8, 26), | ||
| - | description: ' | + | description: 'Rob, Andrew<br>Final chloride assay showed convincing degradation of chloroacetate and DCA using the parts we submitted to the iGEM HQ, also showed that our inducible-promoter systems failed to assemble correctly, instead containing just the constitutive promoter (Ptet). Andrew found further evidence for this by PCR screening.<br><br>Cleaned and packed up a lot of stuff in the lab. ' |
}, | }, | ||
{ | { | ||
title: 'Wikifreeze', | title: 'Wikifreeze', | ||
start: new Date(2013, 8, 27), | start: new Date(2013, 8, 27), | ||
| - | description: ' | + | description: 'Everyone<br>Ohh no!! Wikifreeze! ' |
}, | }, | ||
{ | { | ||
title: 'Hong Kong Jamboree Preparation', | title: 'Hong Kong Jamboree Preparation', | ||
start: new Date(2013, 8, 30), | start: new Date(2013, 8, 30), | ||
| - | description: ' | + | description: 'Everyone<br>Preparing our poster and presentation for HK. ' |
}, | }, | ||
{ | { | ||
title: 'Hong Kong Jamboree Preparation', | title: 'Hong Kong Jamboree Preparation', | ||
start: new Date(2013, 9, 1), | start: new Date(2013, 9, 1), | ||
| - | description: ' | + | description: 'Everyone<br>Preparing our poster and presentation for HK. ' |
}, | }, | ||
{ | { | ||
title: 'Hong Kong Jamboree Preparation', | title: 'Hong Kong Jamboree Preparation', | ||
start: new Date(2013, 9, 2), | start: new Date(2013, 9, 2), | ||
| - | description: ' | + | description: 'Everyone<br>Preparing our poster and presentation for HK. ' |
}, | }, | ||
{ | { | ||
title: 'Hong Kong Jamboree', | title: 'Hong Kong Jamboree', | ||
start: new Date(2013, 9, 4), | start: new Date(2013, 9, 4), | ||
| - | description: ' | + | description: 'Everyone<br>Practice presentation until the early hours. ' |
}, | }, | ||
{ | { | ||
title: 'Hong Kong Jamboree', | title: 'Hong Kong Jamboree', | ||
start: new Date(2013, 9, 5), | start: new Date(2013, 9, 5), | ||
| - | description: ' | + | description: 'Everyone<br>Presentations all day! ' |
}, | }, | ||
{ | { | ||
title: 'Hong Kong Jamboree', | title: 'Hong Kong Jamboree', | ||
start: new Date(2013, 9, 6), | start: new Date(2013, 9, 6), | ||
| - | description: ' | + | description: 'Everyone<br>Won the Best Human Practices and Best Experimental Measurement Approach! ' |
}, | }, | ||
{ | { | ||
title: 'Planning post-HK labwork', | title: 'Planning post-HK labwork', | ||
start: new Date(2013, 9, 9), | start: new Date(2013, 9, 9), | ||
| - | description: ' | + | description: 'Rob, Andrew<br>Designing gBlocks and primers in Ho Chi Minh airport for labwork before MIT.' |
}, | }, | ||
{ | { | ||
title: 'Ordering new gBlocks', | title: 'Ordering new gBlocks', | ||
start: new Date(2013, 9, 10), | start: new Date(2013, 9, 10), | ||
| - | description: ' | + | description: 'Rob, Andrew<br>Convincing our supervisor to purchase the new gBlocks and primers we need. ' |
}, | }, | ||
{ | { | ||
title: 'Finding dhlB', | title: 'Finding dhlB', | ||
start: new Date(2013, 9, 11), | start: new Date(2013, 9, 11), | ||
| - | description: ' | + | description: 'Hugh, Shuravi<br>Transformation of more clones with pSB1C3-dhlB ligation mixture. PCR Screen for correct orientation of dhlB from non-directional ligation. ' |
}, | }, | ||
{ | { | ||
title: 'Isolating dhlB', | title: 'Isolating dhlB', | ||
start: new Date(2013, 9, 12), | start: new Date(2013, 9, 12), | ||
| - | description: ' | + | description: 'Andrew, Rob<br>Plasmid prep of promising clones containing pSB1C3-dhlB.' |
}, | }, | ||
{ | { | ||
title: 'Confirming dhlB', | title: 'Confirming dhlB', | ||
start: new Date(2013, 9, 13), | start: new Date(2013, 9, 13), | ||
| - | description: ' | + | description: 'Andrew<br>Digest confirmation of length and concentration of pSB1C3-dhlB.' |
}, | }, | ||
{ | { | ||
title: 'Cloning dhlB', | title: 'Cloning dhlB', | ||
start: new Date(2013, 9, 14), | start: new Date(2013, 9, 14), | ||
| - | description: ' | + | description: 'Rob<br>Cloning of the promoter Ptet (PCR product) into pSB1C3-dhlB.' |
}, | }, | ||
{ | { | ||
title: 'Confirming expression of dhlB', | title: 'Confirming expression of dhlB', | ||
start: new Date(2013, 9, 15), | start: new Date(2013, 9, 15), | ||
| - | description: ' | + | 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', | title: 'Further confirmation', | ||
start: new Date(2013, 9, 16), | start: new Date(2013, 9, 16), | ||
| - | description: ' | + | description: 'James<br>Chloride assay to confirm chloroacetate degradation. Woo! We’ve got dhlB isolated and it works!' |
}, { | }, { | ||
title: 'gBlocks arrived!', | title: 'gBlocks arrived!', | ||
start: new Date(2013, 9, 17), | start: new Date(2013, 9, 17), | ||
| - | description: ' | + | description: ' Rob, Andrew<br>gBlocks for p450, aldA and tetR arrived. Gibson Assembly and transformation. |
' | ' | ||
}, { | }, { | ||
title: 'Gibson transformation #1', | title: 'Gibson transformation #1', | ||
start: new Date(2013, 9, 18), | start: new Date(2013, 9, 18), | ||
| - | description: ' | + | description: 'Andrew<br>Checked plates after Gibson Assembly - no colonies on tests and colonies on negative controls. Suspect bad plates. Remade plates and re-transformed Gibson reaction product. ' |
}, | }, | ||
{ | { | ||
title: 'PCR screening', | title: 'PCR screening', | ||
start: new Date(2013, 9, 19), | start: new Date(2013, 9, 19), | ||
| - | description: ' | + | description: 'Rob<br>Checked plates again - better! Set-up a huge PCR screen for all Gibson constructs - aldA, p450 and tetR. Retransformed p450 Gibson reaction product to generate more clones for screening. ' |
}, | }, | ||
{ | { | ||
title: 'Plasmid prep', | title: 'Plasmid prep', | ||
start: new Date(2013, 9, 20), | start: new Date(2013, 9, 20), | ||
| - | description: ' | + | description: 'Rob, Andrew, Shuravi<br>More screening. Plasmid prep of promising clones. ' |
}, | }, | ||
{ | { | ||
title: 'Cloning', | title: 'Cloning', | ||
start: new Date(2013, 9, 21), | start: new Date(2013, 9, 21), | ||
| - | description: ' | + | 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. ' |
}, | }, | ||
{ | { | ||
title: 'Waiting for growth', | title: 'Waiting for growth', | ||
start: new Date(2013, 9, 22), | start: new Date(2013, 9, 22), | ||
| - | description: ' | + | description: 'Andrew<br>Transformation plates hadn’t grown sufficiently for screening. ' |
}, | }, | ||
{ | { | ||
title: 'Screening clones', | title: 'Screening clones', | ||
start: new Date(2013, 9, 23), | start: new Date(2013, 9, 23), | ||
| - | description: ' | + | description: 'Rob, Hugh, Andrew<br>Patching and lysing cells from transformation plates for PCR screen and pH-chloroacetate screens. No efficient cloning for whole pathway, only for individual parts with tetR-inducible promoter system. Set-up chloride assay for degradation of DCA by clones containing tetR-p450 and tetR-aldA-dhlB-dhlA.' |
}, | }, | ||
{ | { | ||
title: 'Chloride assay', | title: 'Chloride assay', | ||
start: new Date(2013, 9, 24), | start: new Date(2013, 9, 24), | ||
| - | description: ' | + | description: 'Rob, Andrew<br>Performed chloride assay. Appears there is reasonable degradation of DCA by p450. |
' | ' | ||
}, | }, | ||
| Line 469: | Line 469: | ||
title: 'Acetaldehyde growth inhibition', | title: 'Acetaldehyde growth inhibition', | ||
start: new Date(2013, 9, 25), | start: new Date(2013, 9, 25), | ||
| - | description: ' | + | description: 'Rob, Andrew<br>Set-up an experiment to confirm aldA expression through growth inhibition of control E. coli in LB-acetaldehyde.' |
}, | }, | ||
{ | { | ||
title: 'Characterising new parts', | title: 'Characterising new parts', | ||
start: new Date(2013, 9, 26), | start: new Date(2013, 9, 26), | ||
| - | description: ' | + | 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.' |
} | } | ||
] | ] | ||
Revision as of 05:12, 28 October 2013
