Difference between revisions of "Team:Queens Canada/Notebook"

 
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Latest revision as of 01:11, 17 September 2015

LAB NOTEBOOK

On this page we have documented our lab work and have divided it all into three components, by the construct being generated:

AFP-E coil

Circularized Antifreeze Protein

Scaffold-K coil

Antifreeze Protein - E coil

DAY TASK OUTCOME
June 1st Plated E. coli with Type III AFP donated by Dr. Davies and incubated overnight Very few Type 3 AFP colonies
June 2nd Prepared O/N cultures of Type 3 AFP
June 3rd Mini-prepped wtAFP - Concentration of DNA measured with NanoDrop - Annealed E-coil oligonucleotides
June 4th Digested wtAFP backbone treated with SAP - wtAFP run on agarose to verify linearization - Performed XhoI digest Unsuccessful: SAP likely degraded backbone too much/reaction allowed to proceed for too long
June 5th Digested wtAFP backbone, treated with SAP
June 8th Ligated annealed E-coil oligos into digested wtAFP plasmid with vector: oligo ratios 2:1, 1:1, and 2:3 - Plated wtAFP cells.
June 9th (-) Control plate had highest colony count; shouldn’t have any - (+) Control had a few colonies - Experimental plates had a few colonies
June 10th Inoculated 10 colonies from the 2:1 plate to ON cultures with Amp
June 11th Colony PCR'd liquid cultures of AFP-Ecoil colonies, and ran 2% agarose gels with PCR products and 100bp DNA ladder Unsuccessful insertion of oligos
June 15th Digests were attempted yet again, then SAP treatment -> transformation Successful: colonies present
June 16th Colony screening was done through colony PCR unsuccessful: colony PCR failed
June 22nd Attempted insertion of oligos once more, this time with gel extraction of bands after XhoI digestion
June 26th Ligation attempt - Ligated cells plated on Amp Only successful plate was wtAFP without E-coil - Seems to be nuclease contamination degrading plasmid
July 2nd Again, digestion-extraction-ligation protocol performed (this time treated vectors qithout SAP). Successful: colonies present,picked MANY to find right one
July 3rd XhoI digest for wtAFP-E coil plate for colony screening Successful: certain colonies appear to have an insert - sent for sequencing to confirm the coil is in the right orientation; successful!
August 4th MTransformed correct AFP-E coil into BL21 cells for protein expression Successful: colonies present
August 11th Made 2L of AFP E-coil cells
August 12th Lysed AFP E-coil cells and kept the sup overnight in the cold room (for ice-affinity purification tomorrow) - ran SDS-PAGE of the TCL/sup/pellet to confirm overexpression/solubility of the AFP-Ecoil protein
August 13th Did one round of ice-affinity purification to confirm that the AFP-E.coil fusion can still bind to ice - ran SDS-PAGE of sup, liquid fraction 1, ice fraction 1 The AFP appeared to bind to ice, but we let the crystal grow too large and a bunch of other proteins were incorporated into the ice fraction too - will do another round of ice-affinity tomorrow to further purify
August 14th Ran a second round of ice-affinity purification, starting with the ice fraction from the first round - Ran an SDS-PAGE of the sup, ice/liquid fractions 1, ice/liquid fractions 2 Successful! It appears that the AFP-E coil bound to ice well in the second ice-affinity purification; much better separation!

Circularized AFP (Icefinity)

DAY TASK OUTCOME
June 1st Re-suspended part BBa_K1362000 and transformed into electro- competent E. coli cells plated on Cam resistance plates BBa_K1362000 red colonies visible
June 2nd Grew 1L culture of BBa_K1362000 in LB for imaging, and prepared ON culture of BBa_K1362000 with Cam
June 3rd Mini-prepped BBa_K1362000 liquid culture - Concentration of DNA measured with NanoDrop
June 9th AFP-extein gene arrived
June 10th Re-suspended DNA order - Performed Golden Gate Assembly using BBa_K1362000 from June 3rd and AFP-extein DNA - Transformed reaction products into TopTen electrocompetent cells - Plated on Cam plates Successful: Plates had more white than red colonies.
June 11th O/N culture prepared from white colonies on plate
June 12th Colony screening of white colonies; digested with NotI to determine if insert was present Successful: Insert DNA appears correct size
June 17th Primers resuspended in dH2O - Prepared samples for sequencing
July 2nd Transformed 2 samples in BL21 electrocompetent cells and plated Succesful: 3 colonies on one of the plates, zero on the other
July 6th Prepared ON culture with one transformed colony
July 7th Prepared liquid culture, grew to OD 0.85 - Induced protein expression at 23 degrees C with 1mM IPTG overnight.
July 8th Protein expression; then attempted ice affinity purification - Ran on SDS-PAGE Unsuccessful: appears to be no protein expressed
July 10th Careful troubleshooting and review of plasmid sequence Identified problem pSB1C3 has no T7 promoter sequence… unable to express protein.
July 14th - 17th Insert containing AFP-linker-extein was PCR amplified PCR successful: amplicon band of correct size is visible
July 22nd Digested both the vector and insert with Not1 and Xba1
July 23rd Digests were then ligated then transformed in electrocompetent Topten E.coli K12 cells. Successful insertion of insert into our promoter-containing vector.
July 28th colonies were chosen, DNA purified, then digested with Not1 and Xba1 to confirm correct insertion size Successful: band of insert is of the correct size.
July 30th - August 3rd miniprepped DNA was containing our insert and T7 promoter was transformed into BL21 cells for expression Successful
August 10th O/N cultures of circAFP samples and AFP wild type
August 11th Grew up 2L cultures and induced protein expression AFP circ sequences confirmed with T7 promoter :D
August 12th Isolated cells from cultures - Lysed small fractions of frozen pellet to test intein reaction at various temperatures Gels show a band below the Type III wild type
August 13th Ran gels for test conditions to determine optimal splicing conditions
August 14th Ran TH assay on small fraction crude lysate supernatant to see if protein was active - Lysed frozen pellets - Also performed ice-affinity purification for the circAFP TH gap of 0.3oC → protein is active!!! - Purification will be determined Monday
August 19th - 20th Purified circAFP using size exclusion - Ran SDS-PAGE Successful: single band present on gel
August 24th-28th Subjected pure circAFP to various temperatures - Ran TH assays on the various circAFP to determine activity and thermostability - Analyzed results, put together bar graph. Successful: circAFP appears to retain about 80% of its activity after exposure to 90 degrees C.
September 15th ice-affinity purification was attempted with circAFP - SDS-PAGE was run successful: gel indicated a completely pure circAFP

Scaffold - K coil

DAY TASK OUTCOME
June 18th T3-10 in pET29b arrived from Baker lab
June 22nd Transformed BL21 cells (electrocompetent) with T3-10 scaffold plasmid on Kan plates
June 23rd Selected colony to make liquid culture
June 24th Prepared 1L LB culture with T3-10, grew to OD ~ 2.0 - Protein expression induced (25 deg, 1mM IPTG)
June 25th Cells lysed and centrifuged to remove pellet - Supernatant run through Ni-column and fractions run on SDS-gel to purify Good expression of scaffold protein
June 29th Ran purified T3-10 on S200 column - Re-suspended protein in new buffer Major peak indicative of trimer - May be some 12-mer (small peak)
June 30th Re-ran S200 column - Incubated sample at RT for 48 hours in new buffer No change in chromatograth
July 13th SDS-gel to verify T33-21 unit sizes from Ni2+ purification Units correct size
July 15th-16th Prepared ON cultures of T3-10 scaffold protein- mini-prepped T3-10
July 17th K coil oligonucleotides arrived
July 20th Re-suspended k-coils in dH2O - XhoI digested plasmids, SAP treated - Agarose gel to check before ligation - Plated T3-10 on Kan plate Unsuccessful: gel inconclusive
July 21st Ran T3-10 mini preps on agarose gel - Prepared ON cultures of T3-10 Plasmid confirmed
July 22nd XhoI digested plasmids, SAP treated - Agarose gel to check before ligation - set up overnight ligations with T3-10 and K coil oligos The gels of the XhoI digest looked better - 2 of the samples were used in parallel with hopes one will ligate correctly
July 23rd O/N ligations were transformed into Top10 cells and plated on Kan plates overnight - 2 negative controls (to check digest/SAP) and 1 positive control (test ligase) were used - insert:vector ratios of 1:1 and 1:3 were used Successful: transformations look good, screening next week!
July 27th-28th Grew O/N cultures of potential T3-10-K coil, miniprepped, and screened with digests with BamHI Successful: some appear to have a BamHI site, some samples sent for sequencing
August 10th Sequences indicate cloning was unsuccessful
August 28th T3-10-K coil sequence arrived
August 2nd-4th Prepared to transform T3-10-K coil into iGEM BioBrick vector pSB1C3 ligated DNA was prepped for shipping

NOTEBOOK

AFP-E coil

Circularized Antifreeze Protein

Scaffold-K coil