Team:Queens Canada/Notebook/Week11



Notebook - Week 11

DateProtocolPeopleDNA (if relevant)Quantities and Parameters (if relevant)Notes on Protocol/ Gel Lanes
Jul-09Digestion/ LigationVictorDigest and Ligate fliC1 and fliC2 into their own plasmids and also try
to ligate them together. Attempt 3 trials for each one:
fliC1 + pSB1C3 ---> 3 trials (EP Digest)
fliC2 + pSB1A3 ---> 3 trials (EP Digest)
fliC1 (ES) + fliC 2 (XP) + pSB1C3 (EP) ---> 3 trials
Should have spare digested plasmids already
Varied Digestion Protocols based on trial;
NEB Buffer 2: 2.5 ul
BSA: 0.5 ul
DNA: 2 ul /5 ul (T1, T2)
H2O: 14 ul / 11 ul
Enzymes based on the cell to the right
pS1BC3, may be strange, do no ligate
any of the fliC C1 to it, especially with the individual parts. Need to PCR more fliC C2 parts
 Agar PlatesAndrewMade 40 Normal LB Plates  
Double terminator 1 & 2
1 mL of culture
Jul-10LigationsVictorLigations + Plasmid Resistance ( 3 ul each)
SmTA T1 + fliC1 ES T1 + C
SmTA T1 + fliC1 ES T2 + C
XP SmTA T1 + fliC1 ES T3 + C
XP SmTA T2 + fliC1 ES T4 + C
XP GFP T1 + fliC1 ES T1 + A
XP GFP T4 + fliC1 ES T2 + A
XP GFP T1 + fliC1 ES T3 + A
XP GFP T4 + fliC1 ES T4 + A
XP fmT T1 + fliC C1 ES T1 + T
XP fmT T1 + fliC C1 ES T2 + T
XP fmT T1 + fliC C1 ES T3 + T
XP fmT T1 + fliC C1 ES T4 + T
fliC C1 T5 + fliC C2 XP T1 + C
fliC C1 ES T1 + fliC C2 XP T2 + C
fliC C1 ES T2 + fliC C2 XP T1 + C
fliC C1 ES T3 + fliC C2 XP T2 + C
fliC C1 ES T4 + fliC C2 XP T1 + A
fliC C1 ES T1 + fliC C2 XP T2 + A
fliC C1 EP T1 + pSB1C3
fliC C2 EP T1 + pSB1C3
fliC C1 EP T2 + pSB1A3
fliC C2 EP T2 + pSB1A3
Ligations (x20 Trials)
Ligase Buffer 2 ul
T4 DNA ligase 1 ul
H20 11 ul
3 ul for each specifc part added (as shown left)
Jul-11DigestionVictor6 Digestions:
1) Med RBS T1, 2) Med RBS T2, 3) Strong RBS T1, 4) Strong RBS T2, 5) fliC C1 (XLI Blue) T1 6) fliC C1 (XLI Blue) T2
Digestions Protocols:
NEB Buffer 2: 2.5 ul
BSA: 0.5 ul
Enzyme 1/2: 0.5 ul/ 0.5 ul
DNA: 2 ul (plasmid), 3 ul (PCR)
H2O: 14 ul / 13 ul
 LigationsVictor8 Ligations:
1) Med RBS T1 + FliC C1 (XL1 Blue) T1
2) Med RBS T1 + FliC C1 (XL1 Blue) T2
3) Med RBS T2 + FliC C1 (XL1 Blue) T1
4) Med RBS T1 + FliC C1 (XL1 Blue) T2
5) Strong RBS T1 + FliC C1 (XL1 Blue) T1
6) Strong RBS T1 + FliC C1 (XL1 Blue) T2
7) Strong RBS T2 + FliC C2 (XL1 Blue) T1
8) Strong RBS T2 + FliC C1 (XL1 Blue) t2
 Primer Dilutions David, BeiniXyIE, ech, fcs, and streptividin ---> 5 ul primer in 45 ul TE.  
 PCRDavid, Beiniech control, 1-5 and MBP control, MBP 1 - MBP 5
(K231000) (4 trials + 1 control)
Thermopol Protocol: (12 trials)
Thermopol: 5 ul
dNTPs: 1 ul
LP: 1.5 ul
RP: 1.5 ul
DNA Template: 5 ul
Vent Pol: 0.5 ul
MgSO4: 1 ul
H2O: 34.5 ul

Program:1. Denaturation (Initial): 95 for 5 mins 2. Denaturation: 98C for 20 seconds 3. Annealing: 50C for 15 seconds 4. Extension: 72 degrees, 30 seconds 5. Repeat step 2-4 for 30 cycles 6. Final extension 72 degrees, 5 minute 7. Cool down 4 degrees, one hour
1) Ladder, 2) MBP C, 3) MBP 1, 4) MBP 2,
5) MBP 3, 6) MBP 4, 7) Ladder, 8) RBS med dig T1, 9) RBS med dig T2, 10) RBS med Control, 11) RBS Strong dig T1 (1 band), 12) RBS strong dig T2 (3 bands), 13) RBS strong control (2 bands), 14) fliC C1 dig T1 (same) 15) fliC C1 dig T2 (same)
 Liquid CulturesBeiniLig SmtA + fliC C1 + pSB1C3 (4 trials) The rest of yesterday's ligations are still
in incubator, no colonies yet.
Jul-12Liquid CulturesBeiniLiquid Cultures of:
LIG fliC C1 + pSB1C3 (2 trials)
LIG fliC C2 + pSB1A3 ( 1 trial)
LIG fliC C1 + fliC C2 + pSB1C3 (4 trials)
LIG med RBS + fliC C1 ( 3 trials)
LIG strong RBS + fliC C1 (4 trials)
 MiniprepsVictorFour trials of SmtA  
 Heat shock transformationAndrewPlate of mGFP.  
 Soft Agar Motility PlatesVictorRefer to Soft Agar Motility Plate protocol.  
Jul-13Heat shock Transformation mgfp  
 Liquid CulturesAndrewmgfp and pSB1A3  
1) RBS + fliC C1 ---> SP T1
2) RBS + fliC C1 ---> SP T2
3) fliC C2 ---> XS T1
4) fliC C2 ---> XS T2
5) Medium Terminator ---> B0015 --> XP T1
6) Medium Terminator ---> B0015 --> XP T2
NEB Digestion Protocol:
2 ul Plasmid DNA
0.5 ul EcoRI HF
0.5 ul SpeI (NEB)
2.5 ul NEB Buffer 2
0.5 BSA
14 H20
 MiniprepBeini, AndrewfliC2 + pSB1A3 (two trials), Strong RBS + fliC 1 (4 trials),
Medium RBS + fliC C1 (3 trials)
1) Med RBS + fliC C1 T2 (SP)
2) Med RBS + fliC C1 T3 (Not1)
3) Strong RBS + fliC C1 T1 (SP)
4) Strong RBS + fliC C1 T2 (Not1)
5)fliC C2 + pSB1A3 T2 (Not1)
NEB Digestion Protocol:
2 ul Plasmid DNA
0.5 ul EcoRI HF
0.5 ul SpeI (NEB)
2.5 ul NEB Buffer 2
0.5 BSA
14 H20
1) Ladder, 2) Med RBS + fliC C1 T2 Control, 3) med RBS + fliC C1 T2 SP DIG, 4) med RBS + fliC C1 T3 Not1 DIG, 5) Strong RBS + fliC C1 T1 Control, 6) Strong RBS + fliC C1 T2 Not1 DIG, 7) Strong RBS + fliC C1 T1 SP DiG, 8) Ladder 1 KB+, 9) fliC C2 + pSB1A3 Control T2, 10) fliC C2 + pSB1A3 T2 Not1 DIG, 11) GFP + pSA1C3 Control, 12) GFP + pSA1C3 dig ES, 13) fmT + pSB1C3 Control, 14) fmT + pSB1C3 ES.
1) fliC C2 T1 MPP [XS]
2) fliC C2 T2 MPP [XS]
3) double terminator 1 T2 [XP]
4) Double terminator 2 T1 [XP]
1) (med RBS + fliC C1 T2) + flIC C2 T2 + Term B0017 T1 [A]
2) (med RBS + fliC C1 T2) + fliC C2 T1 + Term B0015 T2 [A]
3) (strong RBS + fliC C1 T1) + fliC C2 T1 + term B0015 T2 [A]
4) (strong RBS + fliC C1 T1) + fliC C2 T2 + term B0017 T1 [A]
 Heat Shock Transfomation Performed heat shock transformation of the above four ligations. Followed standard protocol, but use 50 ul competent cells and 2 ul plasmid DNA.
Potential error: put antibiotic plates in our oven to "prewarm at 37 C," but later realized it wasn't even on.
Leftover transformed cells are in fridge, ( in green Eppendorf tray, labelled as LIG 1 - 4 with date on the side (13/07)
Jul-14MiniprepKevin, MaggieMinipreps of yesterday's ligations   
 DigestionKevin, MaggieFast Digest:
1) 2ul [Lig 1: mRBS + fliC C + term] + 17 ul MM + 1 ul Not1
1) 2ul [Lig 2: mRBS + fliC C + term] + 17 ul MM. + 1 ul Not1
1) 2ul [Lig 3: Str RBS _ fliC C _ term] + 17 ul MM + 1 ul Not1
1) 2ul [Lig 4: Str RBS + fliC C + term] + 17 ul MM + 1 ul Not1
Fast Digest Protocol: (4 Trials)
Water 15 ul
F.D. Green Buffer 2 ul
1) Ladder (1 KB+), 2) Lig 1, 3) Lig 1 (Not1) --> 4 ul (FD Green), 4) Lig 2,
5) Lig 2 (Not1), 6) 1 KB+, 7) Lig 3, 8) Lig 3 (Not1), 9) Lig 4, 10) Lig 4 (Not1)

Monday July 9th

A lot of lab work was done over the weekend. Big up to Kevin and Maggie because--sound the trumpets--they finally managed to isolate the first constant domain of fliC! Hooray! Saturday’s gel revealed a glimmer of hope in the form of a very faint band hovering around 700 kb for one trial of PCR done with genomic DNA of XL1 Blue. Sunday’s gel sealed the deal-- they performed more trials with XL1 Blue genomic DNA, and distinct bands showed up that matched the length of fliC C1. Now, all we need is a working ligation of this gene to be sure that it is our target. To start things off this morning, Kevin gave us a rundown of the busy week ahead. Victor and Andrew went into the lab to get started on making our gigantic hoard of agar plates that we will be needing for all our future ligations. David also went into the lab to do ligations of fliC C2 into plasmid backbones. Research topics today included xylanase (Phillip), isolation of flagella (Beini), and characterization strategies for heavy metal-binding proteins (David). Phillip fiddled around with the wiki and managed to create a sweet background using a photo of Kingston windmills that Kevin took.

Tuesday July 10th

Today in the lab, we performed lots of ligations and heat shock transformations with different combinations of fmT, SmtA, GFP, fliC C1, and fliC C2. We also disposed of old liquid cultures that have been accumulating, as well as a few bottles of contaminated growth medium. A little too much bleach was poured into our liquid waste so some of us got a little heady. Outside of the lab, Phillip worked on potential BioBrick parts for catalysis, David continued updating his heavy metal inventory, and Kevin worked on SynthetiQ sponsorship. Towards the end of the day, a few of us went outside into the glorious sunshine to take some test shots for our gifs. Phillip and Beini held up a white bristol board background while Andrew tried his best to look beautiful, with Kevin behind the camcorder.

Wednesday July 11th

In the lab today, we went through our box of primers to see which ones haven’t been rehydrated. For those that had miniprep products, we rehydrated them for future PCR. We performed digestions and ligations of fliC C1 to ribosomal binding sites (RBSs) of various strength. Outside of the lab, we worked on SynthetiQ sponsorship, primers to order, and fluoroacetate degradation. During our faculty advisor meeting, we updated the professors on our sponsorship progress, SynthetiQ progress, and potential catalysis enzymes. We also inquired about materials that we would like to use for characterization, such as a Waring blender (for isolation of flagella) and a fluorimeter (for fluorescence characterization).

Thursday July 12th

Today, Andrew managed to get his hands on spectinomycin from another lab, so we could finally inoculate mgfp-5, the only part from our last BioBrick order that contained that antibiotic resistance. We made 14 liquid cultures of yesterday’s ligations, of fliC C1 to strong/medium RBSs, although some of the plates just had a few stray colonies on the edge that probably don’t contain the ligated plasmid. Victor and Andrew made soft agar plates for our motility assays in the future. Outside of the lab, we worked on things like: our next round of primers (Andrew), SynthetiQ paperwork (Kevin), documenting new lab protocols (Victor), wiki design (Phillip), heavy metals (David), and expression of flagella by different E. coli strains (Beini).

Friday July 13th

Out of our 14 ligations that we liquid cultured yesterday, 8 worked, so we miniprepped those. We also tried ligating fliC2 and a terminator into a plasmid containing a RBS + fliC1. Victor, our lab manager, is taking his two-week vacation starting on Monday. David, who is very competent and useful with regards to labwork, will also be away next week. So today, the rest of us spent time becoming knowledgeable about lab things, so that hopefully next week things in the lab can still run smoothly.

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