Team:Lethbridge/Notebook/Lab Work/Group1
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* C0040 & B0015 were ligated together in pSB1A3 following the Ligation Protocol 1. | * C0040 & B0015 were ligated together in pSB1A3 following the Ligation Protocol 1. | ||
* Both ligations were transformed following the Transformation Protocol 1. | * Both ligations were transformed following the Transformation Protocol 1. | ||
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<i>Minipreps of pBAD-rbs-lumazine-dt & K331033-K331035</i> | <i>Minipreps of pBAD-rbs-lumazine-dt & K331033-K331035</i> | ||
* Minipreps of pBAD-rbs-lumazine-dt & K331033-K331035 were made following the Miniprep Protocol (Modified Bench Protocol: QIAprep Spin Miniprep Kit Using Microcentrifuge). | * Minipreps of pBAD-rbs-lumazine-dt & K331033-K331035 were made following the Miniprep Protocol (Modified Bench Protocol: QIAprep Spin Miniprep Kit Using Microcentrifuge). | ||
+ | |||
+ | == June 29th, 2011== | ||
+ | <i>PCR of pBAD-P0440 in pSB1K3 and P0440 was done using PCR protocol </i> | ||
+ | <i>Assembly of R0010 with B0034 in pSB1C3 </i> | ||
+ | <i> Transformation for pBad-RBS and pBad- P0440 into Dh5a E. Coli</i> | ||
+ | == June 30th, 2011== | ||
+ | <i> Ran 1% Agarose Gel from June 29 PCR of pBAD-p0440 and p0440 </i> | ||
+ | |||
+ | |||
+ | =July= | ||
+ | == July 2, 2011== | ||
+ | <i>Picked Colonies and grew overnight </i> | ||
+ | *4 colonies picked from pBad rbs in pSB1T3 | ||
+ | *3 colonies picked from pBad-p0440 | ||
+ | *picked 3 colonies from 2 plates of pBAD-P0440 in pSB1C3 | ||
+ | **1 colony picked from plate A, and 2 colonies from plate B | ||
+ | |||
+ | == July 03,2011== | ||
+ | *None of the 4 colonies of pBAD rbs grew, so they were re-grown in the incubator | ||
+ | *Samples of pBAD-p0440 were mini prepped and stored in team 1 DNA box | ||
+ | *Picked 3 samples of pBad- p0440 in psb1C3 and 3 samples of pBAD p0440 in pSB1K3 | ||
+ | |||
+ | == July 05,2011== | ||
+ | <i> PCR out inserts from July 03, of pBAD-P0440</i> | ||
+ | *PCR out inserts from July 03, of pBAD-P0440 samples using prefix and antisense suffix primers and run on agarose gel at 120V to determine if plasmid contains the correct insert, using po440 as a control. | ||
+ | |||
+ | == July 06,2011== | ||
+ | <i>Repeated July 05, but only for pBad p0440 (2,4)</i> | ||
+ | |||
+ | == July 7th, 2011== | ||
+ | <i>Assembled I13453 with B0034 and into pSB1K3 with J04500</i> | ||
+ | *Assembly done using Restriction, ligation, and transformation techniques | ||
+ | |||
+ | == July 11th, 2011== | ||
+ | <i>Assembly of pLacI-rbs and Lumazine synthase- dt</i> | ||
+ | <i>Plasmid transfer of Ag43</i> | ||
+ | *Assembled pLacI-rbs and Lumazine synthase- dt into destination plasmid pSB1C3, followed restriction digest, ligation, and transformation procedures. | ||
+ | *Plasmid Transfer of Ag43 from pSB1C3 to pSB1K3 using cut sites EcoRI and PstI | ||
+ | *Ran a 1% Agarose Gel using 0.5 TAE buffer solution for parts BBa-J04500 in pSB1AK3, R0040, and Lumazine synthase-Dt to confirm sizes. Used Restriction procedure, cutting with EcoRI | ||
+ | |||
+ | == July 13th, 2011== | ||
+ | <i>Assembly of K331033 and K331035</i> | ||
+ | *Did a restriction and Gel Extraction for K331033 cutting at EcoRI and SpeI, K331035 cutting at PstI and XbaI, and pSB1K3 cutting at EcoRI and PstI | ||
+ | *Ran the gel for 80 minutes at 120Volts | ||
+ | *Used Transformation protocol to Transformed XylE, XylF, XylT, XylK, XylQ, into Dh5a E. Coli cells | ||
+ | |||
+ | == July 14, 2011== | ||
+ | <i>Ligation of K331033 to K3310335 into pSB1K3</i> | ||
+ | *Performed a ligation of K331033 to K3310335 into pSB1K3 and transformation of ligated parts into Dh5a E. Coli. Used Transformation and ligation protocols. Plated culture on Kanmycin plates | ||
+ | |||
+ | ==July 15th, 2011== | ||
+ | <i> Assembly of pLacI –Rbs in pSB1C3 and Lumazine Dt in pSB1AK3</i> | ||
+ | *Assembly of pLacI –Rbs in pSB1C3 and Lumazine Dt in pSB1AK3 using restriction digest and gel extraction protocols | ||
+ | |||
+ | == July 19th,2011== | ||
+ | <i> Assembly of K346007 and J04500 into destination plasmid pSB1C3</i> | ||
+ | *Assembly of K346007 and J04500 into destination plasmid pSB1C3 parts were restricted, ligated and transformed using appropriate protocols. | ||
+ | *Results: all of the plates had significant bacterial growth, will repeat experiment using LB instead of SOC during transformation | ||
+ | |||
+ | == July 20th,2011== | ||
+ | <i>Tried to pre-aliquot restriction</i> | ||
+ | *Created a master-mix from appropriately sized aliquots that can be stored in the -20 freezer. Two mixes were created in the following volumes: | ||
+ | **16micro liters miliQ H2O | ||
+ | **2.5 micro liters Buffer II | ||
+ | **0.5 micro liters BSA 100X | ||
+ | **0.5 micro liters EcoRI | ||
+ | **0.5 micro liters SpeI | ||
+ | **20 micro liters total restriction mixture | ||
+ | |||
+ | == July 25th, 2011== | ||
+ | <i>A Colony PCR was run for Parts K346007 and J04500 to confirm assemblies</i> | ||
+ | * This was done following the colony PCR protocol | ||
+ | |||
+ | == July 26th,2011== | ||
+ | <i>Picking of colonies</i> | ||
+ | *Cells were picked with parts J04500- K346007 in them, for plasmid extraction by miniprep protocol. 23 colonies were picked in total and placed in 5 ml of lb in test tubes, along with 5micro liters of chloramphenicol was added to each test tube. Picked colonies were placed in the shaker overnight at 37 degrees Celsius | ||
+ | |||
+ | == July 27th, 2011== | ||
+ | <i>Assembly of following parts:</i> | ||
+ | *K331033 to K331035 | ||
+ | **J04500 to Lumazine Synthase Dt | ||
+ | **pBad to P0440 | ||
+ | **P0440 to K331033 | ||
+ | **Lumazine synthase dt to pBad | ||
+ | *A restriction was done using the restriction protocol following a Gel extraction protocol. A ligation was performed and contents incubated at room temperature for 30 mins. Controls were run with downstream part being substituted as water. | ||
+ | *Restricted J04500-k346007 with EcoRI, and PstI following restriction protocol, to confirm assembly success, restriction were loaded into a 08% Agarose gel. 23 samples from previous day was loaded into gel. Lanes 16 and 17 showed expected sizes | ||
+ | |||
+ | == July 28th,2011== | ||
+ | <i>Ran a PCR gel for Justin to confirm PCR insert was correct</i> | ||
+ | |||
+ | |||
+ | =August= | ||
+ | == August 1st, 2011== | ||
+ | <i> Three colonies picked for parts J45120 and J45320</i> | ||
+ | *3 colonies picked from plates for J45120 and J45320 and were placed in 5 ml test tubes with LB media and 5 micro liters Amp, and placed in shaker overnight | ||
+ | |||
+ | == August 03rd, 2011== | ||
+ | <i>Restriction of:</i> | ||
+ | **pBad-P0440 in pSB1A2 | ||
+ | **Lumazine Synthase-dt with pBad in pSB1A2 | ||
+ | **K331033 – K331035 in pSB1C3 | ||
+ | **P0440 – K331033 in pSB1A2 | ||
+ | *Confirmation of assemblies by means of Agarose Gel | ||
+ | *Ran Gel at 80 volts for 2 hours | ||
+ | *Conclusion: parts were verified and appear to have been assembled successfully | ||
+ | |||
+ | == August 4th, 2011== | ||
+ | <i>Restriction by protocol of assembling parts :</i> | ||
+ | **J04500-Lumazine synthase-dt out of pSB1AK3 + pbad-P0440 in psb1AK3 | ||
+ | **pBad-P0440 out of pSB1A2 + K331033-K331035 in pSB1C3 | ||
+ | **Lumazine synthase-dt-pBad pSB1A2 + p0440-K331033 in psb1A2 | ||
+ | *J04500 in pSB1Ak3 + Lumazine-synthase-dt-pBad out of pSB1A2 | ||
+ | *P0440-k331033 out of pSB1AK3 + k331035 in pSB1C3 | ||
+ | *Ran a TAE agarose Gel | ||
+ | |||
+ | ==August 11th, 2011== | ||
+ | <i>Assembly of parts:</i> | ||
+ | *pBad-P0440 + K331033-k331035 in pSB1C3 | ||
+ | *Lumazine synthase-Dt-pBad + PlacI in pSB1AK3 | ||
+ | *Lumazine synthase in pSB1C3 + Dt in pSB1A2 | ||
+ | *PlacI in pSB1AK3 + Lumazine synthase in pSB1C3 | ||
+ | *Ran a 1X TAE 1% Agarose Gel at 80Volts for 120 min for Extraction of parts by protocol | ||
+ | |||
+ | == August 12th, 2011== | ||
+ | <i>Assembly of J04500 to Enhanced Lumazine synthase and Enhanced Lumazine synthase to Dt</i> | ||
+ | *Followed protocols for Restriction, Gel Extraction, Ligation and Transformation, | ||
+ | *Cut J04500 in pSB1AK3 at SpeI and PstI, Cut Enhanced Lumazine synthase at XbaI and PstI | ||
+ | *Cut Enhanced Lumazine synthase at EcoRI and SpeI, Cut Dt in pSB1AK3 EcoRI and XbaI. | ||
+ | *Samples were run on a 0.9% Agarose gel in 1X TAE at 100Volts | ||
+ | *Results: Colonies grew from the J04500-Enhanced Lumazine synthase and Enhanced Lumazine synthase – Dt in pSB1AK3 | ||
+ | |||
+ | == August 13th, 2011== | ||
+ | <i>Assembly of PLacI with Agn43 with Dt</i> | ||
+ | <br>Miniprep: | ||
+ | *Dt in pSB1AK3 | ||
+ | *J04500 – Agn43 in pSB1AK3 | ||
+ | *K346007(Agn43) in pSB1C3 | ||
+ | *J04500-Lumazine synthase – Dt pSB1AK3 | ||
+ | Ran a Gel of J04500-Ag43 in pSB1A2, and Agn43 in pAB1C3 | ||
+ | Assembly of pBad-P0440 with K331033-K331035 | ||
+ | *Picked: pBad-P0440 cells, K331033- K331035 in pSB1C3 cells | ||
+ | **left cells to grow overnight in shaker at 37 degrees Celsius | ||
+ | **Picked 6 colonies from Enhanced Lumazine synthase plates from August 12 | ||
+ | |||
+ | ==August 14th,2011== | ||
+ | <i>Mini-prepped:</i> | ||
+ | *pBad –p0440 in pSB1A2 | ||
+ | *K331033-K331035 in pSB1C3 | ||
+ | *Enhanced Lumazine synthase | ||
+ | **2 minipreps of plated colonies of pLAcI-Enhanced Lumazine synthase | ||
+ | **4 minipreps of plated colonies of Enhanced Lumazine synthase- Dt | ||
+ | <i>Restricted:</i> | ||
+ | *1) J04500 in pSB1AK3 cut at SpeI and PstI | ||
+ | *2) Enhanced Lumazine syhtase –Dt in pSB1AK3 cut at XbaI and PstI | ||
+ | *3) J04500 –Enhanced Lumazine synthase in pSB1AK3 cut at EcoRI and SpeI | ||
+ | *4) B0017 in pSB1AK3 cut at EcoRI and XbaI | ||
+ | *5) J04500-Lumazine synthase –dt cut at EcoRI and SpeI | ||
+ | *6) pBad- P0440 in pSB1A2 cut at EcoRI and XbaI | ||
+ | *7) pBad- P0440 in pSB1A2 cut at SpeI and PstI | ||
+ | *8) K331033-K331035 Cut at Xba1 and PstI | ||
+ | *Ran a Gel extraction by protocol | ||
+ | <i>The previously numbered restrictions were ligated as follows:</i> | ||
+ | *1+2 | ||
+ | *3+4 | ||
+ | *5+6 | ||
+ | *7+8 | ||
+ | *Ligations were incubated and transformed then plated on Ampicillin plates following protocols. | ||
+ | *A 1X TBE 1% Agarose gel was successfully run at 100volts for 50 minutes to confirm parts | ||
+ | == August 15th, 2011== | ||
+ | <i>Assembly of Antigen 43 with PLacI and Dt</i> | ||
+ | <i>Followed Protocol for a restrictions of :</i> | ||
+ | *1) J04500 in pSB1AK3 | ||
+ | *2) Agn43 | ||
+ | *3) Agn43 | ||
+ | *4)Dt in pSB1AK3 | ||
+ | *5) J04500-Agn43 | ||
+ | *6) Dt in pSB1AK2 | ||
+ | |||
+ | == August 16th, 2011== | ||
+ | <i>Verify parts by running them on an Agarose Gel</i> | ||
+ | |||
+ | == August 17th,2011== | ||
+ | Miniprep protocol was followed for enhanced lumazine synthase in pSB1C3, all 5 ml of culture was used | ||
+ | Restriction of: | ||
+ | *Enhanced Lumazine synthase dt, J04500 + enhanced lumazine and Enhanced lumazine. | ||
+ | *Restricted parts were run on a gel to confirm parts. Gel ran at 80Volts for 90 minutes | ||
+ | *Enhanced lumazine synthase was confirmed, | ||
+ | *J04500-Enhanced lumazine synthase confirmed | ||
+ | *Enhanced Lumazine synthase –Dt did not confirm | ||
+ | <i>Assembly of J04500-enhanced Lumazine synthase +DT </i> | ||
+ | <i>Did a restriction following protocol of:</i> | ||
+ | *1) J04500 – Enhanced Lumazine synthase out of pSB1AK3 Cut at EcoRI and SpeI | ||
+ | *2) B0017 in pSB1AK3 Cut at EcoRI and XbaI | ||
+ | <b>Ran restricted parts on an Agarose Gel: No DNA was able to be observed outside ladder</b> | ||
+ | |||
+ | == August 18th,2011== | ||
+ | <i>Assembled the complete Lumazine synthase and florescent proteins construct</i> | ||
+ | *Repeat August 17th: Restriction, Gel extraction, Ligation and Transformation | ||
+ | *Did a Restriction following protocol of: | ||
+ | **1) J04500-Enhanced lumazine synthase in pSB1AK3 cut at EcoRI and SpeI | ||
+ | **2) B0017 in pSB1AK3 cut at EcoRI and XbaI | ||
+ | **3) J04500-Lumazine synthase – Dt Cut at EcoRI and SpeI | ||
+ | **4) Dt in pSB1C3 cut at EcoRI and XbaI | ||
+ | *Restrictions were run on an Agarose Gel, only parts 1), and 2) were observed on the Gel | ||
+ | Restrictions and Gel were run again for missing parts; restriction time was decreased; no parts were shown on Gel | ||
+ | |||
+ | == August 20th, 2011== | ||
+ | <i>Assembly of Construct:</i> | ||
+ | *J04500- Lumazine synthase-dt-pBad-P0440- K331033-K331035 | ||
+ | *The following protocols were used to assemble this construction: Restriction, Gel Extraction, Ligation and Transformation | ||
+ | <i>Restriction of:</i> | ||
+ | *1) J04500-Lumazine synthase-dt cut out of pSB1AK3 at EcoRI and SpeI | ||
+ | *2) pBad- P0440-K331033-K331035 in pSB1C3 cut at EcoRI and XbaI | ||
+ | *Restrictions were run on a 1% Agarose 1X TAE Gel for purpose of extraction | ||
+ | *DNA did not show on Gel | ||
+ | |||
+ | == August 21st,2011== | ||
+ | <i>Assembly of construct:</i> | ||
+ | *J04500-Lumazine synthase dt-pBad-P0440-K331033-K331035 | ||
+ | *Repeated previous day’s work. The parts had been confirmed by sequencing | ||
+ | *Picked Colonies from plate for parts J04500-Enhanced lumazine synthase in pSB1AK3 and grew overnight in LB for purpose of Glycerol stocks | ||
+ | *Gel of parts worked and a successful Gel extraction was performed. Extracted parts 1) and 2) from August 20th that were ligated together | ||
+ | *Ligated parts were transformed into Dh5alpha and plated on appropriate antibiotic selected plates | ||
+ | |||
+ | == August 22, 2011== | ||
+ | <i>Picked J04500-Enhanced Lumazine synthase colonies from plated construct</i> | ||
+ | <b>Assembly of Constructs:</b> | ||
+ | **1) XylE cut at EcoRI and SpeI | ||
+ | **2) S04261 Cut at EcoRI and XbaI | ||
+ | **3)K331007 cut at EcoRi and XbaI in pSB1C3 | ||
+ | **4) mms6 cut at EcoRI and SpeI | ||
+ | **5) K3301007 in pSB1C3 cut at SpeI and PstI | ||
+ | **6) mms6 Cut at XbaI and PstI | ||
+ | **7) K331008 in pSB1C3 cut at SpeI an dPstI | ||
+ | **8) mms6 cut at XbaI and PstI | ||
+ | **9) K331009 in pSB1C3 cut at SpeI and PstI | ||
+ | **10) mms6 cut at XbaI and PstI | ||
+ | **11) K331012 in pSB1C3 cut at SpeI and PstI | ||
+ | **12) mms6 cut at XbaI and PstI | ||
+ | **13) S04261 pSB1AK3 cut at EcoRI and XbaI | ||
+ | **14) mms6 cut with EcoRI and SpeI | ||
+ | *Parts were run on an Agarose Gel for Gel extraction | ||
+ | *Gel was successful and a Gel Extraction was performed. | ||
+ | <i>Following parts were ligated together as per protocol</i> | ||
+ | *1 + 2 | ||
+ | * 3 + 4 | ||
+ | *5 + 6 | ||
+ | * 7 + 6 | ||
+ | * 9 + 6 | ||
+ | * 11 + 6 | ||
+ | *14 + 4 | ||
+ | <b> Ligations were transformed via protocol into Dh5 alpha, plated, and left in incubator overnight</b> | ||
+ | |||
+ | *Performed a Concentration of Enhanced lumazine synthase from Sephacryl 400 Chromatography | ||
+ | *Pooled fractions at 35, 36 ,37 | ||
+ | *Vivaspin 20, 3000 mwco reviled with 17.5 ml dd milliQ H20, spun at 4000Xg for 25 minutes | ||
+ | *The column was then washed with 10 ml of sodium phosphate buffer, at 4000Xg for 40 minutes | ||
+ | *7.5ml of Fractions 35,36,37,were loaded onto column and concentrated to 1ml by Centrifuge at 4000XG at 4Degrees Celsius for about an hour in 5 minute increments | ||
+ | *Concentrations to be determined photometrically and concentration must be determined and protein visualized Via SDS page gel | ||
+ | |||
+ | ==August 28th, 2011== | ||
+ | <i>Over expression of complete construct</i> | ||
+ | *Overnight culture grown by Justin, 12ml of overnight culture, of E. Coli Dh5alpha J04500-K249002- B0017- I13453-P0040-K331033-K331035, in the pSB1C3 Vector, was inoculated in 4 x 500ml LB flasks, with 500micro liter of Chloramphenicol and grown up to OD600 of 0.0978 | ||
+ | |||
+ | == August 29th, 2011== | ||
+ | *Ran SDS page of August 28th, overexpression, SDS sample preparation protocol was followed | ||
+ | *The SDS page was run at 80Volts for 15 mins and 180Volts for 45mins | ||
+ | *Stained in Coomassie blue for 30 mins and distained over night | ||
+ | |||
+ | ==August 31, 2011== | ||
+ | <i>Assembly of:</i> | ||
+ | *J04500-Enhanced Lumazine synthase cut out of pSB1AK3 at EcoRI and SpeI ligated to B0015 cut at EcoRI and XbaI in pSB1AK3 | ||
+ | *Ptet-rbs in pSB1A2 cut at SpeI and PStI Ligated to XylE - arginine tag cut at XbaI and PstI. | ||
+ | <i>Followed Restriction, Gel Extraction protocols</i> | ||
+ | **Note: J04500 –Enhanced Lumazine synthase and Xyle – agrinine tag –dt did not Cut out of plasmid. B0015 in pSB1AK3, and Ptet-rbs in pSB1A2 were found around the right sizes | ||
+ | |||
+ | |||
+ | =September= | ||
+ | == September 3rd, 2011== | ||
+ | <i>Repeated August 31 assembly</i> | ||
+ | |||
+ | == Sept 14th,2011== | ||
+ | <i>Show how effective BamHI is in degrading E.coli DH5α Genomic DNA</i> | ||
+ | *2 overnight cultures were picked in 50 ml flasks with 50micro liters of Ampicillin and were inoculated with BamHI and pLacI from Glycerol stock | ||
+ | *16ml of pLacI from overnight culture were used to inoculate 250ml of Lb with 250micro liters of Ampicillin to bring it to an OD600 of 0.118 | ||
+ | *11.2 ml of BamHI overnight culture was used to inoculate 250ml of LB to an OD600 of 0.100 | ||
+ | |||
+ | == September 17th, 2011== | ||
+ | <i>Repeat, the August 14th BamHI experiment due to the spectrophotometer being inaccurate</i> | ||
+ | == September 19th, 2011== | ||
+ | <i>Isolation of genomic DNA from BamHI cells by following the Qiagen Blood and Tissue Kit protocol for isolating genomic DNA of gram-negative bacteria </i> | ||
+ | |||
+ | == September 21, 2011== | ||
+ | <i>Overnight culture of full construct (lumazine synthase – K331033 – K331035, K331033 and K331035) for FRET experiments</i> | ||
+ | *Selected cells were induced with arabinose. | ||
+ | *All cells showed growth after 15 hours in shaker at 37 degrees Celsius. | ||
+ | |||
+ | == September 22, 2011== | ||
+ | <i>Continued work from September 21</i> | ||
+ | *Spun down cells and re-suspended in LB media and checked Optical Density of 1 ml of culture and brought them to 1 OD | ||
+ | *Signs of FRET but nothing confirmed | ||
+ | **Note: This is something we will be working on continuously up until Boston |
Latest revision as of 03:32, 29 September 2011
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