Team:Hong Kong HKU/extra/diary

June 28th, Friday
Long Amp PCR + Cloning
Cargo Team: (Only KO genome available)
Native Ko_ppk Amplified > Gel Purified (49ng/ul) > TA vector Ligation+Transformation > Spread Plate 1st step Signal Ko_ppk Am
Surface Team:
plified > Gel Purified (weak) > 2nd PCR
Native EutS Amplified > Gel Purified (25ug/ul) > TA vector Ligation+Transformation > Spread Plate
His_EutS Amplified > Gel Purified > 2nd PCR
Flag_EutS Amplified > Gel Purifed > 2nd PCR
Ko native ppk and Ko sigppk 1st PCR product.



June 29th, Saturday
Long Amp PCR + Cloning, continuous PCR and TA vector transformation.



June 30th, Sunday
His Euts TA colonies for culture.
Redo transformation of native EutS and Flag EutS.
Continuous PCR of Tf_ppk1 and Ko_ppk1.



July 1st, Monday
Miniprep of HisEutS and Ko_ppk1 TA clone culture and Digestion test.
Overnight ligation of Native Ko_ppk1, Native Tf_ppk1, pLacHisEutS, Sig_Koppk1(Weak), Flag_EutS



July 2nd, Tuesday
Mass Digestion of His_EutS.
Continuous PCR and gel purification.
Miniprep for EutS Topo conlonies.
4 Transfromation (Topo Clone) : Native Ko_ppk1, Native Tf_ppk1, pLacHisEutS, Sig_Koppk1(Weak), Flag_EutS



July 3rd, Wednesday
Continuous PCR of Ko_sigppk, His_EutSMNLK and Flag_EutSMNLK.
Mass Digestion of native EutS.
Transformation: His_Eut into K525998 (Cm Plate); TF_sigppk into Topo (Amp+Xgal Plate); Flag_EutS into Topo (1),(2)



July 4th, Thursday
MiniPrep and Digestion Test: Native Ko_ppk1, Native Tf_ppk1, Sig_Koppk1
Transformation: NativeEutS into 525 (1,2) Cm Plate; Native Tfppk, Koppk, sig_koppk
Prepare DH10B Competent Cells.



July 5th, Friday
Miniprep and Digestion Test: HisEutS 525, FlagEutS topo, Tf_sigppk topo.
2nd Round PCR for His,Flag EutSMNLK



July 6th, Saturday
A very Tough Weekend (6-8/7)
PCR Native Ko, Native Tf, Sig Tf and Sig Ko was ligated in to Topo Vector.
Many White Colonies were observed. (Also with a few Blue Colonies)
Very HAPPY about the result and picked colonies for culture.
Culture Two Times and Found out the growth rate was abnormally Slow and the Culture was not cloudy and contain
fillament like clusters.
Maybe the ppk gene might be toxic to cell?<< Wrong
Maybe the shaker speed and temperature not correct? << Wrong
WE have Fungus/other bacteria Comtamination!!!

Hints:
Colonies morphology>>very white and not transparent and moist compared to Ecoli colonies
Culture grow well (withou filament clusture) in 32C.
Can grow in Wrong Antibiotics. (Topo vector is Kana resistant, I use Cm to test>Still grow)
Tried to miniprep on tube with less clusters. No Plasmid.
Right Now We don't Know the source of contamination.

Overview:
What we have:
We have EutS, His EutS, Flag EutS in Topo Vector. Positive Digestion Pattern.
Send for sequencing on 9/7.
To avoid comtaminantion.
Will Redo Transformation to create a new stock.

But Right now, we have:
HisEutS 1,2 glycerol stock and streak plates.
FlagEutS 1,2 glycerol stock and streak plates.
(picked colonies from plates and tested postive, but cannot tell whether there is comtamination)
EutS Retransformed plates and glycerol stock.
Previous EutS, His_EutS, Flag_EutS in to 525 vector failed. Will try again.

But we still don't have any ppk in vector!! But have succeed in PCR (But result random)

Cannot Amplify EutSMNLK from K311004. Will send K311004 for sequencing.



July 7th, Sunday


July 8th, Monday


July 9th, Tuesday
Miniprep and Digestion Test of Tf native ppk, Tf sigppk and Ko sigppk.
Redo ligation and transformation of Ko native ppk, Ko sigppk and Tf sigppk.



July 10th, Wednesday
PCR of Ko native ppk, Tf sigppk, Ko sigppk
Tranformation of 4 kind of ppk1.
Mass Digestion of Tf native ppk plasmid 1,2
Overnight culture of Tf native ppk1.



July 11th, Thursday
Colony PCR for EutS, HisS, FlagEutS and Tfppk into 525 backbone ligation Positive colonies are cultured.
MiniPrep of three Tf native ppk
Making competent cells.
Ligation of Tf native DNA (XbaI/ PstI) with 525 vector.
Transformation of Tf sigppk.



July 12th, Friday
1st round PCR of KO sigppk1
Miniprep of EutS, HisS, FlagEutS and Tfppk in 525 backbone.
Digestion Test



July 13th, Saturday
Digestion test.
Picked EutS525 4, HisEutS525 1, FlagEutS525 1, Tfppk525 2 plasmid transformed into BL21
Colony PCR of sigppk.



July 14th, Sunday
Check plates (Ko native ppk and Ko sigppk 1st PCR product) → pick 34 colonies for colonies PCR → Run gel, two from ko native ppk showed 2100bp. → Culture these two colonies in 5ml LB overnight.
Digestion of Tf sigppk plasmid (XbaI and PstI) and purify 2100bp tf sigpkk DNA → Overnight ligation with 525 vector.
Ligation of Ko sigppk PCR product into topo vector → Trasformation



July 15th, Monday
Miniprep and Digestion Test: Ko native ppk.
Transformation of Tf sigppk 525 vector (into Cm plate), Ko sigppk topo vector (into Kan plate)
Pick two colonies from Tf sigppk stock plate and culture overnight.



July 16th, Tuesday
Check plates (Tf sigppk 525, Ko sigppk topo) and colonies PCR, culture 6 colonies.
Digestion Ko native ppk overnight.



July 17th, Wednesday
Miniprep and digestion test of Topo Ko sig 1,2,3 and Tf sigppk 525 1,2,3.
Digestion overnight (Ko sigppk 3, Tf sigppk1 and K525998 plasmids)
Gel Purification of digestion product.



July 18th, Thursday
Run gel and purification of Digestion Product.
Ligation of Ko native ppk and Tf sigppk to new 525 vector, transformation.
Transformation of Ko native ppk/525 into DH10B, spread plate.
Pick two colonies from Ko native ppk and one colony from Ko sigppk stock plates and culture overnight.



July 19th, Friday
Colonies PCR of Ko native ppk 525, Tf sigppk 525.
MiniPrep of yesterday's culture. ( Ko native ppk topo and Ko sigppk topo)
Pick 2 colonies from Euts native525, 2 from Flag Euts525, 2 from His Euts525, 1 from Tf native ppk525 and culture overnight.



July 20th, Saturday
Antibiotics Mistake, all culture failed. Redo culture again.



July 21st, Sunday
MiniPrep and digestion test of Ko native ppk 525, Tf sigppk525.
Transformation of Ko native ppk 525 vector into Bl21 and spread plates.
Culture Tf native ppk 525 BL21 overnight. Culture 4 tubes of K311004 DH10B overnight.



July 22nd, Monday
Check plates: No colonies. Thus redo transformation of Ko native ppk 525 vector into Bl21.
Miniprep of K311004 DH10B, concentrate into one plasmid purification kit. Get a high conc. plasmid sample (110.4ng/ul)
IPTF induction of Tf native ppk Bl21, culture overnight.



July 23rd, Tuesday
Run PAGE of induced Tf native ppk Bl21 cell lysate and control, coomassie blue staining.



July 24th, Wednesday
As yesterday's result was not ideal, redo PAGE and coomassie blue staining.



July 25th, Thursday
Check the coomassie blue staining result: A 500kDa band (referring to the sequencing result, it might be a premature stop codon. normal ppk: 78kDa)
PCR and Purification: Ko native ppk(Template: Ko genomic DNA); Tf sigppk1 2nd PCR(template: WY's Tf sigppk1 1st PCR product); Ko sigppk 1st PCR (template: new making Ko native ppk)
PCR test: plasmid Sigppk1 topo 1, Plasmid sigppk1 topo 2
Culture Ko sigppk topo 1,2,3 overnight.
Culture Tf native ppk bl21 ovenight.



July 26th, Friday
IPTG inductionof Tf native ppk bl21(10, 20).
Miniprep and Digestion test of Ko sigppk topo 1,2,3.
Run gel of Ko sigppk 3 PCR product.
Ligation of Tf sigppk and Ko native ppk into topo vector and transformation
Culture Euts native, Flag, His Bl21 overnight.



July 27th, Saturday
Run PAGE of Tf native ppk bl21, coomassie blue staining.
IPTG induction of Euts Native, Flag, His.
Check plates. (White colonies, Contamination?)



July 28th, Sunday
Colony PCR of TF sigppk and Ko native ppk topo DH10B
Run PAGE of Euts Native, Flag, His Bl21. Coomassie Blue staining.
Transformation of Tf sigppk topo and spread plate.



July 29th, Monday
Check plate of Tf sigppk topo, colony PCR.
Miniprep of Ko native ppk topo and Digestion test.
IPTG induction of Tf native ppk.
Transformation of Euts native, flag, his; Tf sigppk 1, 5 (all in 525 vector) into BL21 and spread plates.


July 30th, Tuesday
Run gel of digestion product, purification. Ligation into 525 vector.
Run PAGE of Tf native ppk and coomassie blue staining.
Miniprep of Tf sigppk topo.
Culture Euts native, flag, his; Tf sigppk 1, 5 BL21 colonies overnight for IPTG induction.


July 31st, Wednesday
IPTG induction of Euts native, flag, his; Tf sigppk 1, 5
Digestion of Tf sigppk topo and digestion test. Ligation into 525 vector.
Transformation of Ko native ppk 525 into DH10B and spread plates.



August 1st, Thursday
Run PAGE gel of Euts native, flag, his; Tf sigppk 1, 5 and Coomassie blue staining.
Transformation of Tf sigppk 525 into DH10B and spread plate.
Colony PCR of Ko native ppk 525 DH10B
1st Round PCR of Tf sigppk.


August 2nd, Friday
Miniprep and digestion test.
Colony PCR.


August 3rd, Saturday
Miniprep of Tf sigppk, Ko native ppk in DH10B ,tf native ppk 5, native EutS, Flag EutS, His EutS in BL21. Digestion test.
Try IPTG induction again using a new condition.
Transformation of Tf sigppk 2,5, 7 in BL21.


August 4th, Sunday
PAGE of EutS native, Flag EutS and HisEutS.


August 5th, Monday
Transform BBa_E1010 plasmid into DH50, Cm plates


August 6th, Tuesday
Pick colonies for culture


August 7th, Wednesday
Miniprep E1010 plasmid. Perform Digestion test
Design primer for Signal fused GFP



August 8th, Thursday
Mass digest of RFP(P+X)


August 9th, Friday
Primers arrived Gel purify digested RFP, ligation to 525 vector, transformation into DH10B


August 10th, Saturday
PCR ppk for commercial vector(adding tag for purification) PCR sig-RFP for detecting localization Transformation of KO525 (4, 19) into bl21 Transformation of ligation product (RFP into 525) 1st PCR of Sig Ko


August 11th, Sunday
Colony PCR of RFP-525 plates, culture positive colonies
Gel Purify 1st PCR SigKo, 2nd PCR
Transform Ko525(4), Ko525(19) into DH5a for DNA storage
Overnight Culture BL21: KO525(4)(19),TFsig(2),TF(6)(7)(8)

Transform Ko Topo 1, Ko Topo 2, Ko Topo 3, Tf Topo A, Tf topo B into BL21 and spread plates and grow overnight. Second PCR sig-RFP, Purified and digest using S+P overnight
Digest 525948 Vector using S+P overnight
Ligatation of sig 2nd PCR product and TOPO



August 12th, Monday
Miniprep RFP525 (2,4,5,6)>>Digestion test>>Transform (4) into BL21
Gel purify 2nd SigKo, SigRFP >> Ligate into T-vector>> Transform DH5a
Transform Pet28a (kana R) into DH5a to get vector
Transform K311004,HisSMNLK,FlagSMNLK into BL21
Streak HisSMNLK (1a),FlagSMNLK(4b) DH5a to get DNA
Culture NativeEutS, HisS, FlagS 525 to get DNA
IPTG induce all BL21 (Try 0.5mM iptg, 16C)
Bacteria culture of Ko Topo 1,2,3, and Tf Topo A,B
Ligation of SpeI and PstI cut K525998 and sig RFP
Colony PCR of Ko 1 (11,12), Ko 3(6,7,8,9,10),Tf(1,2,3,4,5)


August 13th, Tuesday
Prepare Cm plates
Run gel of colony PCR products from 12-Aug
Miniprep of bacteria culture TfB (3,4,5) and KO (2,4,5), digestion test of miniprep products with NdeI and NotI Transformation of Sig RFP Topo and 525 into DH10B and Spread Plate
Harvest All Induced BL21, Run SDS-PAGE


August 14th, Wednesday
Bacteria culture of RFP Topo and RFP 525.
Redo transformation of RFP Topo into DH10B.



August 15th, Thursday
Miniprep of bacteria culture from 14/8.


August 16th, Friday
Send out 4 plasmids for DNA sequencing.
Digestion of Ko topo 2,4 Tf and Pet(Commercial vector).



August 17th, Saturday
1st PCR SigKO


August 18th, Sunday
Gel purification of mass digestion, ligation of sigRFP into525
2nd PCR SigKO, purified, ligate into Tvector


August 19th, Monday
Colony PCR for SigKO inTvector, SigRFP in 525
Culture SigKO in Tvector
Miniprep SigRFP in 525


August 20th, Tuesday
Miniprep SigKO in Tvector, send for sequencing
Miniprep SigRFP in 525, Digestion Test
Digestion test of TFsig+HisS/EutS/FlagS/HisK
Result: SigKO-tvector all correct, pick 2 for mass digest, TfSig-hisK2,5, Tfsig+His,Eut,FlagS all correct, SigRFP525 all correct
Transform in to BL21: TfsigHisK, SigRFP525, Tfsig_HisS,FlagS,EutS


August 21st, Wednesday
Bacteria culture of Tf pET28a(t). Double digestion test of Ko pET28a(t).


August 22nd, Thursday
Miniprep of Tf pET28a(t) and digestion test.


August 23rd, Friday
Run gel of digestion product from 22/8. Spead plates of KO pET28a(t) 2, 4.


August 24th, Saturday


August 25th, Sunday
Malachite Green phosphate assay


August 26th, Monday
Run gel of Digestion Product and purification.


August 27th, Tuesday
Preparing BL21 competent cells.
Malachite Green phosphate assay.
Bacteria culture.


August 28th, Wednesday
Miniprep and digestion test.


August 29th, Thursday
Bacteria culture. Ligation and transformation into DH10B.


August 30th, Friday
Miniprep and Digestion Test.


August 31st, Saturday
Bacteria culture.


September 1st, Sunday
Miniprep and digestion test. Transformation.


September 2nd, Monday
Overnight culture of RFP collections


September 3rd, Tuesday
IPTG induction


September 4th, Wednesday
Observe under fluorescent microscope, didn’t see localization RFP


September 5th, Thursday


September 6th, Friday
Miniprep Kop ET 28a(t) DH10B and digestion product. Digestion and purification of pET28a(t) vector. Transformation. Bacteria culture.


September 7th, Saturday


September 8th, Sunday
Miniprep and Digestion test.


September 9th, Monday
His-tagged protein purification.


September 10th, Tuesday



September 11th, Wednesday
His-tagged protein purification. PAGE of the purified protein, coomassie blue staining.


September 12th, Thursday


September 13th, Friday


September 14th, Saturday
Overnight culture KO collections


September 15th, Sunday
IPTG induce KO collections


September 16th, Monday
Harvest cell pellet for polyphosphate extraction


September 17th, Tuesday
DAPI assay, run gel for toluidine blue staining


September 18th, Wednesday
Gel very intensive. Try to load fewer amount and compare with ppk2 treatment


September 19th, Thursday
Run gel for toluidine blue staining


September 20th, Friday
Polyphosphate Assay Phosphate Assay SigRFP localization of Eut MCP