Team:Austin UTexas LASA/Notebook
Notebook
Sept 1 - Sept 9, 2016
Week 1: Got aliquots of HpaBC from lab, grew up parts (Hpab, HpaC, pp2551) and digested them to check lengths.
Oct 3- Oct 7, 2016
Week 1: Did PCR to get HpaB out of the HpaBC lab plasmid, sequence verified ppddc in BTK001 backbone, sequence verified pp2551 and HpaC, made glycerol stocks of sequence verified parts
Oct 10 - Oct 14, 2016
Week 2: Ordered HpaBC operon from IDT, grew up pEM7 [BTK156], did Gibson assembly of HpaBC in YTK001 entry vector and grew it up
Oct 17 - Oct 21, 2016
Week 3: Split parts into different teams, each team working on assembling and sequence verifying individual parts into entry vecotrs[ppddc, HpaBC pEM7, pp2551]
Oct 24 - Oct 28, 2016
Week 4: Grew up and miniprepped HpaBC, and pp2551, but transformation of HpaBC did not work.
Nov 1- Nov 3, 2016
Week 1: Did Gibson assembly, transformed and miniprepped ppddc, pEM7, and HpaBC, found that Gibson assembly for HpaBC did not work
Nov 6 - Nov 10, 2016
Week 2: Redid Gibson assembly for HpaBC, realized we were cloning our parts into YTK001 [plasmid vector]
Nov 13 - Nov 17, 2016
Week 3: Transformed, miniprepped, made glycerol stocks of HpaBC. Did a gibson assembly of ppddc.
Nov 20 - Nov 24, 2016
Week 4: Transformed pp2551, transformed Gibson product of pEM7, transformed and miniprepped ppddc gibson assembly.
Dec 1- Dec 8, 2016
Week 1: pp2551 plates had no colonies, streaked with glycerol stocks of HpaBC, redid transformation of pp2551
Dec 11 - Dec 15, 2016
Week 2: Transformation of HpaBC, ppddc plates had no colonies, redid Gibson assembly for ppddc
Dec 18 - Dec 22, 2016
Week 3: miniprepped and made glycerol stocks of HpaBC, got very low concentrations for the minipreps, looked into origin of the backbone (BTK001). Transformed gibson reaction of ppddc, plate had no colonies.
Dec 25 - Dec 29, 2016
Week 4: Digested ppddc and backbone (BTK001) to see if there was a problem in the ori, ran on a gel, showed no insert.
Jan 1- Jan 5, 2017
Week 1: Used Zymo DNA clean up kit to get more concentrated values to send in for sequencing. Established plan for how to proceed with each part.
Jan 8 - Jan 12, 2017
Week 2: Miniprepped pp2551. Designed primers for ppddc pcr to add golden gate overhangs.
Jan 15 - Jan 19, 2017
Week 3: Sent in HpaBC and pp2551 to sequencing. Started ‘lab trainings’ with highschool students new to the club.
Jan 22 - Jan 26, 2017
Week 4: Received primers for ppddc, and diluted them.
Feb 1- Feb 9, 2017
Week 1: Ran a PCR reaction of ppddc to add golden gate overhangs.
Feb 12 - Feb 16, 2017
Week 2: Received and grew up an aliquot of new backbone (YTK001) with ColE origin because the original backbone was giving us trouble with getting high enough concentrations to sequence verify it.
Feb 19 - Feb 23, 2017
Week 3: Miniprepped new backbone (YTK001). Did a PCR clean-up of ppddc.
Feb 26 - Feb 28, 2017
Week 4: Re-ran PCR reaction and purified ppddc because we were running low.
March 1- March 10, 2017
Week 1: Ran a gel electrophoresis on ppddc and HpaBC. Golden gate reaction of ppddc and ytk001, then purified it. Transformed ppddc + ytk001, both green and white colonies (transformation successful).
March 13 - March 17, 2017
Week 2: Miniprepped ppddc + ytk001. Transformed pp2551 from January. Grew up pp2551 for glycerol stocks and Hpabc for miniprep to do a golden gate reaction with ytk001.
March 20 - March 24, 2017
Week 3: Miniprepped HpaBC and nanodropped, but concentrations too low to do a golden gate assembly. Re-did liquid cultures then miniprep of HpaBC.
March 27 - March 31, 2017
Week 4: Figured out/planned how to combine the two separate sensing plasmids (venus cassette and pp2551 cassette). Decided to do multigene assembly using a new backbone. Looked into addition of lac operon in the sensing assembly.
April 1- April 7, 2017
Week 1: n/a, people out for school.
April 10 - April 14, 2017
Week 2: Did a BsaI golden gate reaction for venus cassette plasmid (VCP). Used a backbone (BTK402) with a pMMB67EH origin, ppddc promoter, rpoc terminator, and ConL1 and ConRE connectors.
April 17 - April 21, 2017
Week 3: Dilutions of each part of the venus cassette plasmid. Grew them up to have enough of our own stock.
April 24 - April 31, 2017
Week 4: Did a BsaI golden gate reaction for production assembly (HCP) with a CP25 promoter, HpaBC, rpoc terminator, conLS and conRE connectors.
May 1- May 5, 2017
Week 1: Miniprepped each of the cassette plasmid parts. Transformed VCP golden gate reaction. Did a ___golden gate reaction for pp2551 cassette plamisd (PCP).
May 8 - May 12, 2017
Week 2: Made glycerol stocks of VCP. Transformed HCP, PCP, and VCP. No growth on plates. Looked into why golden gate didn’t work. Found out the low-copy origin is not efficient, and z-comp cells we used for transformation are less efficient.
May 22 - May 26, 2017
Week 3: Redid golden gate reaction of HCP, PCP, and VCP using EcoRI enzyme. When we spun down liquid cultures, the cells were all red. Re-picked colonies (white) from the HCP and VCP plate because PCP plate only had red colonies. Did a miniprep of HCP and VCP.
May 29 - May 31, 2017
Week 4: Digested and ran gel electrophoresis of HCP and VCP. No insert seen.
June 1- June 9, 2017
Week 1: Out for Vacation
June 12 - June 16, 2017
Week 2: Out for Vacation
June 19 - June 23, 2017
Week 3: Tried golden gate reaction of pp2551, negative results. Ran production assembly, gel looked good. Decided to focus on sensing assembly.
June 26 - June 30, 2017
Week 4: Out for Vacation
July 1- July 7, 2017
Week 1: Tried digesting all parts necessary for golden gate reaction of pp2551’s cassette plasmid, digest did not run properly. Received evpp2551 from Simon (backup), PCRed to add golden gate overhangs.
July 10 - July 14, 2017
Week 2: Went back to retrying golden gate reaction of pp2551 after eliminating several possible places of error, didn’t work. Put evpp2551 into a golden gate vector.
July 17 - July 21, 2017
Week 3: Digested all parts necessary for golden gate reaction of pp2551, several oddities with smaller pieces. Decided to move on to possibilities of gibson assembly of sensing assembly. Started working on Human Practices and Wiki in earnest.
July 24 - July 31, 2017
Week 4: Designed primers for overhangs of CP25, pp2551, and Venus for gibson assembly. Retried a golden gate reaction to assemble the pp2551 cassette plasmid, grew up, but did not seem to have any colonies with the correct insert (all colonies were red instead of white from the rfp dropout). Looked into a new backbone to clone the cassette assemblies into.
July 1- July 7, 2017
Week 1: Tried digesting all parts necessary for golden gate reaction of pp2551’s cassette plasmid, digest did not run properly. Received evpp2551 from Simon (backup), PCRed to add golden gate overhangs.
July 10 - July 14, 2017
Week 2: Went back to retrying golden gate reaction of pp2551 after eliminating several possible places of error, didn’t work. Put evpp2551 into a golden gate vector.
July 17 - July 21, 2017
Week 3: Digested all parts necessary for golden gate reaction of pp2551, several oddities with smaller pieces. Decided to move on to possibilities of gibson assembly of sensing assembly. Started working on Human Practices and Wiki in earnest.
July 24 - July 31, 2017
Week 4: Designed primers for overhangs of CP25, pp2551, and Venus for gibson assembly. Retried a golden gate reaction to assemble the pp2551 cassette plasmid, grew up, but did not seem to have any colonies with the correct insert (all colonies were red instead of white from the rfp dropout). Looked into a new backbone to clone the cassette assemblies into.
July 1- July 7, 2017
Week 1: Tried digesting all parts necessary for golden gate reaction of pp2551’s cassette plasmid, digest did not run properly. Received evpp2551 from Simon (backup), PCRed to add golden gate overhangs.
July 10 - July 14, 2017
Week 2: Went back to retrying golden gate reaction of pp2551 after eliminating several possible places of error, didn’t work. Put evpp2551 into a golden gate vector.
July 17 - July 21, 2017
Week 3: Digested all parts necessary for golden gate reaction of pp2551, several oddities with smaller pieces. Decided to move on to possibilities of gibson assembly of sensing assembly. Started working on Human Practices and Wiki in earnest.
July 24 - July 31, 2017
Week 4: Designed primers for overhangs of CP25, pp2551, and Venus for gibson assembly. Retried a golden gate reaction to assemble the pp2551 cassette plasmid, grew up, but did not seem to have any colonies with the correct insert (all colonies were red instead of white from the rfp dropout). Looked into a new backbone to clone the cassette assemblies into.
August 1- August 4, 2017
Week 1: Ordered primers for sequence verification and gibson assembly of cassette plasmid parts. Redid PCR reactions of parts (CP25, evpp2551, venus, backbone) to add gibson overhangs). Decided to try using a new backbone with lacI gene. Digested and ran gels to check lengths of PCRed parts. All parts looked fine. Got in contact with Power for Parkinson’s
August 7 - August 11, 2017
Week 2: Did golden gate reactions for backbone (ColE + lacI). Ran a gibson reaction of parts (CP25, evpp2551, venus, backbone). Started looking into testing the promoter pddc background. Visited and helped out at Power for Parkinson’s (Public Engagement)
August 21 - August 18, 2017
Week 3: Sent in the gibsoned multigene assembly and backbone (btk524) for sequencing. Transformed Venus cassette plasmid into BL21 DE3s. Sequencing did not work.
August 28 - August 31, 2017
Week 4: Digested the sensing multigene assembly.
September 1- September 8, 2017
Week 1: Ran the digests on a gel. Restreaked and grew up plates of Venus cassette plasmid. Ran a pre-experiment with Venus Casette plasmid with a plate reader.
September 11 - September 15, 2017
Week 2: Sent sensing multigene assembly for sequencing again using PCR primers.
September 18 - September 22, 2017
Week 3: Could not assemble the backbone with a ColE origin. Decided to stick with a p15A origin and LacI.
September 25 - September 29, 2017
Week 4: Digested and ran Venus cassette plasmid and evpp2551 cassette plasmid. Extracted and purified gel. Looked into testing plasmids of another sensing plasmid from the lab that has a similar construct.
October 1- October 6, 2017
Week 1: Conducted experiment on the already made sensing mulitigene assembly from the lab. Did a golden gate assembly of the evpp2551 cassette plasmid.
October 9 - October 13, 2017
Week 2: Sent evpp2551 cassette plasmid for sequencing. After sequence verifying, moved onto a multigene assembly.
October 16 - October 20, 2017
Week 3: Reached out to igem mentor for help. Ordered designed primers for Venus cassette plasmid and evpp2551 cassette plasmid. Did two step digest for backbone (pET28), venus cassette, evpp2551 cassette. Gel extracted them and ligated. Grew up and prepared liquid cultures for part characterization. Acquired several different promoters along with the biobrick promoter to characterize.
October 23 - October 31, 2017
Week 4: Did experiments for part characterization and sensing plasmid.
Task Division
Lab: Ashley, Anna, Eli, Anusha, Anika, Shreya, Sumin, Shruti, David, Christian
Wiki: Anika, Shreya, Ashley, Anna
Human Practices: David, Shruti, Anna
Public Relations: Sumin, Christian
