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Team:Lethbridge/Notebook/Lab Work/Justin
From 2011.igem.org
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| - | <image src="https://static.igem.org/mediawiki/2011/ | + | <image src="https://static.igem.org/mediawiki/2011/1/14/Justina.jpg" height="400px"/> |
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=Week 3 (May 16-22)= | =Week 3 (May 16-22)= | ||
| - | == | + | ==Monday== |
<p>Check to see if Group 1 has successfully assembled biobricks by running samples on a 1% agarose gel followed by analyzing. | <p>Check to see if Group 1 has successfully assembled biobricks by running samples on a 1% agarose gel followed by analyzing. | ||
</p> | </p> | ||
| Line 141: | Line 135: | ||
==Sunday== | ==Sunday== | ||
| - | <p>Express BamH1 restriction endonuclease in E. coli DH5α in comparison to wild type E. coli DH5α that were not induced. BamH1 restriction endonuclease was induced with arabinose. Optical density measurements were taken and a growth curve of both cultures was generated. | + | <p>Express BamH1 restriction endonuclease in E. coli DH5α in comparison to wild type E. coli DH5α that were not induced. BamH1 restriction endonuclease was induced with arabinose. Optical density measurements were taken and a growth curve of both cultures was generated. Also samples after induction were taken to run on an SDS-PAGE. |
</p> | </p> | ||
=Week 4 (May 23-29)= | =Week 4 (May 23-29)= | ||
| - | == | + | ==Monday== |
<p> | <p> | ||
| + | Justin recuperated after working through the weekend. | ||
</p> | </p> | ||
==Tuesday== | ==Tuesday== | ||
| - | <p> | + | <p>A 15% SDS-PAGE of samples taken from the BamH1 over-expression was ran. Gel looks promising ☺ |
</p> | </p> | ||
==Wednesday== | ==Wednesday== | ||
<p> | <p> | ||
| + | Parts that were constructed recently were PCR amplified. | ||
</p> | </p> | ||
==Thursday== | ==Thursday== | ||
| - | <p> | + | <p>An agarose gel was ran of the PCR products to determine which parts had the correct size. Parts that had an appropriate size range were sent for sequencing. |
| + | Parts sent for sequencing: | ||
| + | *1) ROO10-BOO34 | ||
| + | *2) K249002-BOO15 | ||
| + | *3) K331033-K331035 | ||
| + | |||
</p> | </p> | ||
==Friday== | ==Friday== | ||
| + | <p>Justin assisted team #1 | ||
| + | </p> | ||
| + | |||
| + | ==Saturday== | ||
| + | <p> Time was spent researching, and assisting teams that spent the weekend working in lab. | ||
| + | </p> | ||
| + | |||
| + | =Week 5 (May 30-31 and June 1-5)= | ||
| + | |||
| + | ==Monday== | ||
<p> | <p> | ||
| + | Colones were picked from Morgan's (group 2) plates from 05/27/11 and set up for an overnight culture. | ||
</p> | </p> | ||
| + | ==Tuesday== | ||
| + | <p> | ||
| + | Overnight cultures from May 30, 2011 were mini-preped using the QIAGEN spin column method. Then the DNA was PCR amplified and ran on a 1% agarose gel. Parts that were sent for sequencing were: | ||
| + | *1) pBAD-P0440 | ||
| + | *2) K249002-B0015 | ||
| + | </p> | ||
| + | <p> Assembly of 3 BioBricks! | ||
| + | *1) pBad-rbs + lumazine-dt | ||
| + | *2) K331033 + K331035 | ||
| + | *3) R0010 + B0034 | ||
| + | **--> We want to have pLacU-rbs-lumazine-dt, but since we have pbad and rbs we will use above. This will result in expressing lumazine synthase ASAP :)</p> | ||
| + | <p> Dustin helped out with construction of parts</p> | ||
| + | |||
| + | ==Wednesday== | ||
| + | <p>Justin helped out team 3 | ||
| + | </p> | ||
| + | |||
| + | ==Thursday== | ||
| + | <p>Justin figured out what we need to order for lab, and compiled sequencing results.</p> | ||
| + | <p> Set up a PCR of Assemblies from May 31 to run on an agarose gel of constructs. | ||
| + | </p> | ||
| + | |||
| + | ==Friday== | ||
| + | <p>Ran PCR products from June 2 on an agarose gel and retransformed DNA in DH5α | ||
| + | </p> | ||
| + | <p>Transform DNA that will be sent for sequencing on Monday (suspected BioBricks). Parts that will be transformed: | ||
| + | *1) K249002 + B0015 | ||
| + | *2) pBAD + P0040 | ||
| + | *3) R0010 + B0015 | ||
| + | *4) K249002 + B0015 | ||
| + | *5) K331033 + K331035 | ||
| + | </p> | ||
==Saturday== | ==Saturday== | ||
| + | <p> All plates from transformations had some white colonies. Ranged from a lawn of white colonies to very few white colonies. White colonies were picked off each plate and a overnight culture was setup.</P> | ||
| + | |||
| + | ==Sunday== | ||
<p> | <p> | ||
| + | Dee ran an agarose gel of several parts (Ryan pulled these out of glycerol stocks) to determine which parts should be sent for sequencing. | ||
</p> | </p> | ||
| - | =Week 5 ( | + | =Week 6 (June 6-12 )= |
| - | == | + | |
| + | ==Monday== | ||
| + | <p> Cleaning the lab up a bit. So much work on our project has been done so far. Go team! | ||
| + | |||
| + | </p> | ||
| + | |||
| + | ==Tuesday== | ||
| + | <p> Helping Ryan figure out some PCR issues | ||
| + | </p> | ||
| + | |||
| + | ==Wednesday== | ||
| + | <p> Reading synthetic biology papers and preparing for SynBio 5.0! Also getting some autoclaving done. | ||
| + | </p> | ||
| + | |||
| + | |||
| + | ==Friday== | ||
| + | <p>Attending Journal club at the University of Lethbridge. Got some great ideas | ||
| + | </p> | ||
| + | |||
| + | ==Saturday== | ||
| + | <p> Mini-preped 5 samples/ colonies that Nathan assembled (pBAD-rbs-lumazine-dT) using QIAGEN mini-prep method. Then the parts were PCR amplified, and will later be restricted and ran on a agarose gel.</P> | ||
| + | ==Sunday== | ||
| + | <p> PCR products from June 11, 2011 were ran on a 1.5% agarose gel to confirm their size</p> | ||
| + | |||
| + | =Week 7 (June 13- 19)= | ||
| + | |||
| + | <p>Justin left for ISMOS-3 on June 12, 2011. Had a great time on June 13,2011 listening to the keynote Steve Larter for the University of Calgary on "Microbes or Mass Transport -What are the Real Barriers to Production Scale Microbial Gasification of Oil or Coal to Produce Methane or Hydrogen in Subsurface Reservoirs?" The conference was very informative applied microbiology and molecular biology research that is occurring in the oil sands.</p> | ||
| + | |||
| + | <p>Then Justin left for SynBio 5.0 held at Stanford University. "I Attended SynBio 5.0, an international conference held this year at Stanford University in Palo Alto, California. At this conference, fellow team members and I got to see some of the leading experts in the field of synthetic biology and some of the amazingly creative research they are doing. Considering my love for iGEM, this conference gave me a whole new passion for this years iGEM competition." - Justin Vigar (transcribed by Harland Brandon)</p> | ||
| + | |||
| + | =Week 8 (June 20-26)= | ||
| + | |||
| + | ==Monday== | ||
<p> | <p> | ||
| + | Planed out experimental protocol for over-expressing CFP with an agranine (10) tag (K331033) and purification using cation exchange | ||
| + | |||
</p> | </p> | ||
==Tuesday== | ==Tuesday== | ||
| + | <p>Gathered materials needed for over-expresion and purification of CFP with an arg tag. Made buffers for purification. | ||
| + | </p> | ||
| + | |||
| + | ==Wednesday== | ||
<p> | <p> | ||
| + | Setup overnight culture of CFP with arg tag (K331033) for over-expression. | ||
</p> | </p> | ||
| + | ==Thursday== | ||
| + | <p>Over-expressed CFP with arg tag (K331033). Cell were frozen for purification down the road. | ||
| + | </p> | ||
| + | =Week 9 (June 26-30 )= | ||
| + | ==Monday== | ||
| + | <p> Calibrating pipettes, filling tip boxes and autoclaving. And mopping floor, Woo! | ||
| + | |||
| + | </p> | ||
| + | |||
| + | ==Tuesday== | ||
| + | <p> Aided team # 2 | ||
| + | </p> | ||
| + | |||
| + | ==Wednesday== | ||
| + | <p> PCR amplified | ||
| + | *1) lumazine synthase-dt from glycerol stocks | ||
| + | *2) R0010-B0034 assembly | ||
| + | *3) R0010 in pSB1A2 (control) | ||
| + | *4) K331035 | ||
| + | *5) K331033 | ||
| + | *6) pSB1A3 (J04450) | ||
| + | *7) lumazine synthase | ||
| + | </p> | ||
| + | <p> Then Ran PCR products on a 2% agorose gel.</p> | ||
| + | <p> constructs that were the correct size were: | ||
| + | *1) Lumazine synthase | ||
| + | *2) lumazine synthase-dt | ||
| + | **the rest were undetermined. | ||
| + | |||
| + | ==Thursday== | ||
| + | Attempted purification of CFP arg (K331033) tagged protein. There are some issues that need to be solved.</p> | ||
| + | |||
| + | ==Friday== | ||
| + | <p>Problem solving and working on wiki. | ||
| + | </p> | ||
| + | =Week 10 (July 5-10)= | ||
| + | |||
| + | ==Tuesday== | ||
| + | <p>Clone J04500, pBAD-B0034, and K346007 into E. coli DH5α | ||
| + | </p> | ||
| + | ==Wednesday== | ||
| + | <p> Pick colonies of cloned parts, July 6, 2011, and started overnight culture | ||
| + | </p> | ||
| + | ==Thursday== | ||
| + | <p>Express BamH1 and show that growth is hindered; Minipreped cell cultures that were grown overnight to determine quality/quantity/size of DNA of certain parts; Assebled pLacI-RBS with lumazine-dT. Also a growth curved of a induced (arabinose) and un-induced culture were generated from optical density measurements. | ||
| + | </p> | ||
| + | <p> After 8 hours dilutions were made from the induced and un-induced cultures and plated.</p> | ||
| + | <p><html><a href="https://2011.igem.org/Team:Lethbridge/Results#Results_7"><font color="blue">Results</font></a></html></p> | ||
| + | |||
| + | ==Friday== | ||
| + | <p> Mini-prep cell cultures that were grown overnight, by Nathan, to determine the quality/quaintly/ size of DNA of parts: | ||
| + | *1) J04500 | ||
| + | *2) pBAD-B0034 | ||
| + | *3) K346007 </p> | ||
| + | |||
| + | <p>Mini-preped samples were restricted then ran on a 1% agarose gel. We were unable to detect DNA for pBAD-B0034 on the agarose gel. All other parts were visible and had an acceptable size.</p> | ||
| + | ==Saturday== | ||
| + | <p> Continue assembly on our lumazine BioBrick, which I'm really excited about. I think we will be able to construct our whole massive construct with lumazine synthase and the fluorescent proteins. It will be so cool if we can show that the construct will express the proteins and work as a true interchangeable part!!!</p> | ||
| + | <p> Assembly of pLacI-rbs (J04500) with lumazine.</p> | ||
==Sunday== | ==Sunday== | ||
| + | <p> Transformation of yesterdays work! Transformed lumazine construct into E. coli DH5α.</p> | ||
| + | |||
| + | =Week 12 (July 17-24)= | ||
| + | |||
| + | ==Wednesday== | ||
| + | <p>Determine if xyl transformations were successful and if K346007 was successfully transferred from pSB1C3 to pSB1K3. This was done by restricting the mini-preps and running a 1% agarose gel. It looks like we might have successfully assembled for the gel: | ||
| + | *pSB1A3 promotor-rbs-xylJ-dt | ||
| + | *pSB1A3 promoter-rbs-xylE-dt | ||
| + | * pSB1A3 promoter-rbs-xylF | ||
| + | *pSB1A3 promoter-rbs-xylk | ||
| + | *pSB1A3 promoter-rbs-xylQ | ||
| + | *pSB1A3 promoter-rbs-xylE-pSB1A3 promoter-rbs-xylQ | ||
| + | *pSB1A3 promoter-rbs-xylE-pSB1A3 promoter-rbs-xylF | ||
| + | *pSB1A3 promoter-rbs-xylE-pSB1A3 promoter-rbs-xylJ | ||
| + | *pSB1A3 promoter-rbs-xylE-pSB1A3 promoter-rbs-xylK | ||
| + | *J04450 pSB1C3 | ||
| + | *J04450 psb1k3 | ||
| + | *K346007 psB1K3 | ||
| + | These will need to be sequence to confirm they are the parts we believe them to be. | ||
| + | </P> | ||
| + | |||
| + | ==Thursday== | ||
| + | <p>Today Team Justin determined if the assembly of R0010 (placI-rbs) and IC346007 (Agn43) was successful. Note: Boris did this assembly 07/20/11, see team 1 book for details. Plates that were transformed with parts do have white colonies :). These white colonies were selected and colony PCR was performed using the Prefix and Suffix primers. | ||
| + | </p> | ||
| + | |||
| + | ==Friday== | ||
| + | <p> Samples from colony PCR (yesterday) were ran on a 1% agarose gel. Amplification was successful! However, no inserts of right size are present. | ||
| + | </P> | ||
| + | |||
| + | =Week 13 (July 25-31)= | ||
| + | ==Monday== | ||
<p> | <p> | ||
| + | Assemble K331035-K331033 and R0010-luma-dT</p> | ||
| + | First what happened is the BioBricks were restricted out of the plasmids. Then to determine the concentration of DNA after the restrictions a 1% agarose gel was ran. | ||
| + | |||
| + | <p>The team has made so much progress, we are all very excited for Indianapolis! We had such a successful team meeting:) GO TEAM! | ||
</p> | </p> | ||
| - | < | + | |
| + | ==Tuesday== | ||
| + | <p> Determine DNA concentration of J04500, K331033, K331035, and lumazine synthase dt. Team JV (Justin and Mr. Vigar) concluded that DNA concentration was too low for the spectrophotometer to read. However, the DNA was still assembled and transformed (by Ben).</p> | ||
| + | <p> Then K331033 was ligated with K331035, and J04500 with lumazine-dt. Constructs were transformed into E. coli DH5α cells | ||
| + | </p> | ||
| + | |||
| + | ==Wednesday== | ||
| + | <p>Colonies were counted from yesterdays transformation. Both constructs resulted in white colonies --> We'll perform colony PCR on these samples see July 27, 2011 | ||
| + | </P> | ||
| + | |||
| + | |||
| + | =Week 14 (August 1-7)= | ||
| + | ==Tuesday== | ||
| + | First day in a three day experiment to isolate enhanced lumazine synthase from ''E. coli'' BL21(DE3) cells. Set up overnight culture, checked plasmids, made buffers. | ||
| + | |||
| + | ==Wednesday== | ||
| + | Second day, lumazine synthase is overexpressed. Open cells and run SDS-PAGE gel. Purification of lumazine synthase. | ||
| + | |||
| + | |||
| + | ==Saturday== | ||
| + | Concentrated lumazine synthase. SDS-PAGE done to confirm this. | ||
| + | |||
| + | =Week 15 (August 8-14)= | ||
| + | ==Monday== | ||
| + | Buffers made for chromatography. Two assemblies are ran for lumazine + inverter and inverter + tagged fluorescent proteins. | ||
| + | |||
| + | ==Friday== | ||
| + | Colony PCR of assemblies. Confirm inverter + tagged fluorescent. | ||
| + | |||
| + | =Week 16 (August 15-21)= | ||
| + | ==Tuesday== | ||
| + | Picked colonies from plates to get more plasmids to assemble with. | ||
| + | |||
| + | =Week 17 (August 22-28)= | ||
| + | ==Tuesday== | ||
| + | Assemblies of lumazine + inverter and fluorescent proteins. | ||
| + | |||
| + | ==Wednesday== | ||
| + | Transformation of yesterdays assemblies. | ||
| + | |||
| + | ==Thursday== | ||
| + | Helped out team 2 while Ryan is gone by testing assemblies of mms6 with polypetide signal tags and working with Dustin to assemble xylE with arginine polypeptide signal tag. | ||
| + | |||
| + | ==Friday== | ||
| + | Mms6 testes negative on an agarose gel. | ||
| + | |||
| + | =Week 18 (August 29 - September 4)= | ||
| + | ==Monday== | ||
| + | Prepping cellular samples for electron micrograph imaging. | ||
| + | |||
| + | ==Tuesday== | ||
| + | Finished prepping samples for electron micrograph imaging. | ||
| + | |||
| + | ==Friday== | ||
| + | Images of cells expressing lumazine synthase taken. | ||
| + | |||
| + | |||
| + | =Week 20 (September 12-18)= | ||
| + | ==Tuesday== | ||
| + | LB cultures made from glycerol stocks. | ||
| + | |||
| + | ==Wednesday== | ||
| + | Mini-prepped cultures and assembled the isolated parts into psb1c3. | ||
| + | |||
| + | ==Thursday== | ||
| + | Continued assemblies from Wednesday. | ||
| + | |||
| + | ==Friday== | ||
| + | Continued assemblies from Wednesday and Thursday. | ||
| + | |||
| + | =Week 21 (September 19-25)= | ||
| + | Prepared for aGEM in Edmonton | ||
| + | |||
| + | =Week 22 (September 26-28)= | ||
| + | ==Monday== | ||
| + | Sent parts to the registry. | ||
| + | |||
| + | ==Tuesday== | ||
| + | Sent parts to the registry with Ryan. | ||
| + | |||
| + | ==Wednesday== | ||
| + | Gone to Calgary to give a presentation on the synthetic biology and the future of the oil sands. | ||
Latest revision as of 03:50, 29 September 2011
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