June 22, 2011
PCR of pBAD-P0440 in pSB1K3
- pBAD-P0440 was PCRed following the PCR Protocol.
June 23, 2011
Confirmation Gel of pBAD-P0440 (PCR)
- pBAD-P0440 (PCR) was restricted following the Restriction Protocol 4.
- A gel was made following the 1% Agarose Gel Protocol (100mL).
- The restrictions were loaded onto the gel with a 1kb ladder.
- The gel ran at 100V for 90 minutes.
- The gel was then stained in Ethidium Bromide for 10 minutes.
- TABLE? PICTURE?
Overnight Cultures of pBAD-rbs-lumazine-dt & K331033-K331035
- Overnight cultures of pBAD-rbs-lumazine-dt & K331033-K331035 grown following the Overnight Cultures Protocol.
June 24, 2011
Minipreps of pBAD-rbs-lumazine-dt & K331033-K331035
- 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
PCR of pBAD-P0440 in pSB1K3 and P0440 was done using PCR protocol
Assembly of R0010 with B0034 in pSB1C3
Transformation for pBad-RBS and pBad- P0440 into Dh5a E. Coli
June 30th, 2011
Ran 1% Agarose Gel from June 29 PCR of pBAD-p0440 and p0440
July
July 2, 2011
Picked Colonies and grew overnight
- 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
PCR out inserts from July 03, of pBAD-P0440
- 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
Repeated July 05, but only for pBad p0440 (2,4)
July 7th, 2011
Assembled I13453 with B0034 and into pSB1K3 with J04500
- Assembly done using Restriction, ligation, and transformation techniques
July 11th, 2011
Assembly of pLacI-rbs and Lumazine synthase- dt
Plasmid transfer of Ag43
- 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
Assembly of K331033 and K331035
- 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
Ligation of K331033 to K3310335 into pSB1K3
- 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
Assembly of pLacI –Rbs in pSB1C3 and Lumazine Dt in pSB1AK3
- Assembly of pLacI –Rbs in pSB1C3 and Lumazine Dt in pSB1AK3 using restriction digest and gel extraction protocols
July 19th,2011
Assembly of K346007 and J04500 into destination plasmid pSB1C3
- 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
Tried to pre-aliquot restriction
- 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
A Colony PCR was run for Parts K346007 and J04500 to confirm assemblies
- This was done following the colony PCR protocol
July 26th,2011
Picking of colonies
- 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
Assembly of following parts:
- 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
Ran a PCR gel for Justin to confirm PCR insert was correct
August
August 1st, 2011
Three colonies picked for parts J45120 and J45320
- 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
Restriction of:
- 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
Restriction by protocol of assembling parts :
- 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
Assembly of parts:
- 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
Assembly of J04500 to Enhanced Lumazine synthase and Enhanced Lumazine synthase to Dt
- 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
Assembly of PLacI with Agn43 with Dt
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
Mini-prepped:
- 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
Restricted:
- 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
The previously numbered restrictions were ligated as follows:
- 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
Assembly of Antigen 43 with PLacI and Dt
Followed Protocol for a restrictions of :
- 1) J04500 in pSB1AK3
- 2) Agn43
- 3) Agn43
- 4)Dt in pSB1AK3
- 5) J04500-Agn43
- 6) Dt in pSB1AK2
August 16th, 2011
Verify parts by running them on an Agarose Gel
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
Assembly of J04500-enhanced Lumazine synthase +DT
Did a restriction following protocol of:
- 1) J04500 – Enhanced Lumazine synthase out of pSB1AK3 Cut at EcoRI and SpeI
- 2) B0017 in pSB1AK3 Cut at EcoRI and XbaI
Ran restricted parts on an Agarose Gel: No DNA was able to be observed outside ladder
August 18th,2011
Assembled the complete Lumazine synthase and florescent proteins construct
- 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
Assembly of Construct:
- J04500- Lumazine synthase-dt-pBad-P0440- K331033-K331035
- The following protocols were used to assemble this construction: Restriction, Gel Extraction, Ligation and Transformation
Restriction of:
- 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
Assembly of construct:
- 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
Picked J04500-Enhanced Lumazine synthase colonies from plated construct
Assembly of Constructs:
- 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.
Following parts were ligated together as per protocol
- 1 + 2
- 3 + 4
- 5 + 6
- 7 + 6
- 9 + 6
- 11 + 6
- 14 + 4
Ligations were transformed via protocol into Dh5 alpha, plated, and left in incubator overnight
- 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
Over expression of complete construct
- 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
Assembly of:
- 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.
Followed Restriction, Gel Extraction protocols
- 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
Repeated August 31 assembly
Sept 14th,2011
Show how effective BamHI is in degrading E.coli DH5α Genomic DNA
- 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
Repeat, the August 14th BamHI experiment due to the spectrophotometer being inaccurate
September 19th, 2011
Isolation of genomic DNA from BamHI cells by following the Qiagen Blood and Tissue Kit protocol for isolating genomic DNA of gram-negative bacteria
September 21, 2011
Overnight culture of full construct (lumazine synthase – K331033 – K331035, K331033 and K331035) for FRET experiments
- Selected cells were induced with arabinose.
- All cells showed growth after 15 hours in shaker at 37 degrees Celsius.
September 22, 2011
Continued work from September 21
- 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
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