Team:Queens Canada/Notebook/Week12

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Notebook - Week 12

DateProtocolPeopleDNA (if relevant)Quantities and Parameters (if relevant)Notes on Protocol/ Gel Lanes
Jul-16Fast DigestionBeini, FaisalFast Digest:
1) 2ul [GFP + pSB1C3] + 17 ul MM + 1 ul Not1
2) 2ul [SmtA + fliC C1 + pSB1C3 T1] + 17 ul MM. + 1 ul Not1
3) 2ul [SmtA + fliC C1 + pSB1C3 T2] + 17 ul MM + 1 ul Not1
4) 2ul [FliC C2 T1] + 17 ul MM + 1 ul Not1
5) 2ul [FliC C2 T2] + 17 ul MM + 1 ul Not1
6) 2ul [FliC C2 + pSB1A3 T2 (2) ] + 17 ul MM + 1 ul Not1
7) 2ul [Strong RBS + fliC C1 T2] + 17 ul MM. + 1 ul Not1
8) 2ul [Med RBS + fliC C1 T2] +17 ul MM + 1 ul Not1
9) 2ul [fliC C1 + pSB1A3 T2] + 17 ul MM + 1 ul Not1
10) 2ul [ fliC C2 T1] + 17 ul MM + 1 ul Not1
Fast Digestion Mastermix (10 trials):
Water 15 ul = 150 ul
10x FD Green Buffer 2 ul = 20 ul
+ Reagents as listed in cell to the left
Gel (All are Not1 digests)
1) Ladder 1 KB+, 2) GFP + pSB1C3, 3) SmTA + fliC C1 + pSB1C3 T1, 4) SmtA + fliC C1 + pSB1C3 T2, 5) fliC C2 T1, 6) fliC C2 T2, 7) Ladder 1 KB+, 8) fliC C2 + pSB1A3 T2 (2), 9) Strong RBS + fliC C1 T1, 10) Med RBS + fliC C1 T2, 11) fliC C1 + pSB1A3 T2, 12) fliC C2 T1 heat denaturing
 PCRAndrew, PhillipPCR for ( 2 trials, 1 control)
mgfp ( BBa_K197018), ech (vanillin) (BBa_K238028), PBrR (MPP trial 1 & 2) (BBa_I721002), ECEP (BBa_E0022), xyiE (BBa_K147002), N-split mCer ( BBa_K323011), C-split mCer (BBa_K323012), fcs (BBa_K238029), streptavidin ( BBa_K283010), Csplit mCitr (BBa_K323055)
Thermopol Protocol:
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
Total: 50 ul

Program:1. Denaturation (Initial): 95 C for 5 mins 2. Denaturation: 95 C for 20 seconds 3. Annealing: 50 C 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
Gel (All NotI Digests)/Actual Bands
1) 1 KB+ Ladder
2) GFP (E0040) + pSB1C3/ ~1500
3) SmtA+fliC1+pSB1C3 T1 / ~2200
4) SmtA+fliC1+pSB1C3 T2/~ 2200
5) fliC C2 (pCR2.1 (synthesis plasmid)) T1/ 700, 4-5000
6)fliC C2 (pCR2.1) T2/ ~2100, ~1800
7) Ladder/ ~1400
8) fliC C2 + pSB1A3/ ~1400
9) Strong RBS fliC C1 T1/ ~2000
10) Med RBS fliC C1 T2/ ~2000
11) fliC C1 + pSB1A3 T2/ ~ 1400
12) fliC C1 T1 Heat Denatured/ ~5000
 PCR GelPhillip, AndrewSmall Gel:
1) Ladder 1 KB+, 2) ech C, 3) ech 1, 4) ech 2, 5) ECFP C, 6) ECFP 1, 7) ECFP 2
Large Gel Note ( lane order is reversed)
1) Ladder, 2) xyIE C, 3) xyIE 1, 4) xyIE 2, 5) PBrR1 C (written as mgfPC), 6) PBrR1 1 (Written as mgfP1), 7) pBrR1 2 (written as mgfp2), 8) Ladder, 9) pBrR2 C, 10) PBrR2 1, 11)PBrR2 2, 12) Csplit mCitr C, 13) Csplit mCitr 1, 14) Cplit mCitr 2.
Large Gel Note
1) Ladder, 2) N-split mCer C, 3) N-split mCer 1, 4) N-split mCer 2, 5) C-split mCer C, 6) C-split mCer 1, 7) C-split mCer 2, 8) Ladder, 9) fcs C, 10) fcs 1, 11)fcs 2, 12) Strep C, 13) Strep 1, 14) Strep 2.
Jul-17PCR PCR of fliC1 using promoter primer  
 Rehydration BBa_B0034 (RBS) (2 trials)Thermopol Protocol: (5 trials)
Thermopol: 5 ul
dNTPs: 1 ul
LP: 1.5 ul
RP: 1.5 ul
DNA Template: 10 ul
Vent Pol: 0.7 ul
MgSO4: 2 ul
H2O: 38.3 ul
Total: 50 ul

Program:Not recorded
Made another tube of 5x XLI Blue DNA
using 10 ul 10X XLI Blue DNA and 40 ul TE
included LP & RP in mastermix
 Heat Shock TransformationAndrewRBS B0034  
Jul-18Bacterial Cell CulturePhillipRBS B0034 T1, T2 Took bottle of 2xTY from Chin Sang's lab
+ 1 box of yellow pipette tips
 Gel Dan, Beini  Gel:
1)Ladder, 2)pFliC C, 3) pFliC 1, 4) pFliC 2, 5) pFliC 3, 6) pfliC 4.
 Fast Digestion/ GelPhillip, BeinifliC1 w/ SpeI (six trials)Fast Digest Protocol:
2 ul DNA fiC1
2 ul 10x FD Buffer Green
1 ul SpeI (FD)
15 ul H20
Gel:
1) Ladder, 2)fliC1 T1 [1], 3) fliC1 T1 [2], 4) fliC1 T2 [1], 5) fliC1 T2 [2], 6) fliC1 T3 [1], 7) fliC1 T3 [2]
 Glycerol StockKevinUsed the Glycerol stock protocol.  
 Fast Digest PCR Product of: GFP, smTa, Gold Binding Protein (GoIB)Fast Digest Protocol:
2 ul DNA fiC1
2 ul 10x FD Buffer Green
1 ul SpeI (FD)
15 ul H20
Done in preparation of PCR overlap extension
 PCR Overlap extensionBeini, FaisalfliCC1 and GFP, smTa, Gold Binding Protein (GoIB)Thermopol Protocol:
Thermopol: 5 ul
dNTPs: 1 ul
LP pfliC1: 1.5 ul
RP insert: 1.5 ul
pfliC1 template DNA: 2.5 ul
insert Template DNA: 2.5 ul
Vent Pol: 0.7 ul
MgSO4: 1 ul
H2O: 28.3 ul
Total: 33.3 ul

Using LP of fliC C1 and RP of respective part.

Program:1. Denaturation (Initial): 95 C for 5 mins 2. Denaturation: 98 C for 20 seconds 3. Annealing: 65 C for 15 seconds 4. Extension: 72 degrees, 60 seconds 5. Repeat step 2-4 for 25 cycles 6. Final extension 72 degrees, 5 minute 7. Cool down 4 degrees, one hour
 
 PCR Phillip, AndrewfliC (four trials one control)Thermopol Protocol:
Thermopol: 5 ul
dNTPs: 1 ul
LP pfliC1: 1.5 ul
RP insert: 1.5 ul
Template DNA: 10 ul
Vent Pol: 0.7 ul
MgSO4: 1 ul
H2O: 28.3 ul
Total: 33.3 ul

Using LP of fliC C1 and RP of respective part.

Program:1. Denaturation (Initial): 95 C for 5 mins 2. Denaturation: 98 C for 20 seconds 3. Annealing: 54 C for 17 seconds 4. Extension: 72 degrees, 60 seconds 5. Repeat step 2-4 for 36 cycles 6. Final extension 72 degrees, 5 minute 7. Cool down 4 degrees, one hour
 
 GelKevin  Gel:
1)1KB+, 2) pFliC C, 3)pFliC 1, 4) pFliC 2, 5) pFliC 3 6)pFliC 4. 7) pFliC1 + GFP C, 8) pFliC 1+ GFP 1, 9)1 KB+, 10) pFliC 1+GFP 2, 11) pFliC 1 + GolBC, 12) pFliC 1 + GoIB1, 13) pFliC 1 + GoIB 2, 14) pFliC 1 + Smt C, 15) pFliC 1 + Smt1
Jul-19Liquid CulturesKevinLiquid Culture of DH5α from last night  
 PCR Overlap Extension FaisalPCR Overlap Extension from yesterday with more plymerase and dNTPs being put in the same PCR tubes. The tubes being done again are: 1) pFliC1 + GFP C, 2) pFliC 1+ GFP 1, 3)1 KB+, 4) pFliC 1+GFP 2, 5) pFliC 1 + GolBC, 6) pFliC 1 + GoIB1, 7) pFliC 1 + GoIB 2Thermopol Protocol:
Thermopol: 5 ul
dNTPs: 1 ul
LP pfliC1: 1.5 ul
RP insert: 1.5 ul
pfliC1 template DNA: 2.5 ul
insert Template DNA: 2.5 ul
Vent Pol: 0.7 ul
MgSO4: 1 ul
H2O: 28.3 ul
Total: 33.3 ul

Using LP of fliC C1 and RP of respective part.

Program:1. Denaturation (Initial): 95 C for 5 mins 2. Denaturation: 98 C for 20 seconds 3. Annealing: 68 C for 15 seconds 4. Extension: 90 degrees, 60 seconds 5. Repeat step 2-4 for 15 cycles 6. Final extension 72 degrees, 5 minute 7. Cool down 4 degrees, one hour
 
 DigestionBeini, PhillipGFP digestion w/ X and SStandard NEB Protocol: Used 2 ul of GFP1 PCR product found in bundle w/ GFP 1,2,4. 
 MiniprepKevinRBS B0034   
 PCR Overlap ExtensionBeini, Phillip Thermopol Protocol:
Thermopol: 2.5 ul
dNTPs: 0.5 ul
GFP Digest w/ XS: 5 ul
fliC2 Digest w/ XS: 5 ul
pfliC1 PCR product: 5 ul
Vent Pol: 0.35 ul
MgSO4: 1 ul
H2O: 5.64 ul
Total: 25 ul

Using LP of fliC C1 and RP of respective part.

Program:1. Denaturation (Initial): 95 C for 5 mins 2. Denaturation: 95 C for 20 seconds 3. Annealing: 60 C for 15 seconds 4. Extension: 72 degrees, 30 seconds 5. Repeat step 2-4 for 15 cycles 6. Final extension 72 degrees, 5 minute 7. Cool down 4 degrees, one hour
 
 PlatingKevinTop 10 using sealed competent cells,  
 Motility Assay TestKevin Innoculated the center of two motility plates using
two colonies from the DH5α culture plate which were prewarmed for five minutes. They were innoculated by stabbing colony and then stabbing center of plate
 
 Gel on PCR Overlap ExtensionFaisal Gel:
1) Ladder, 2) pFliC1 + GFP C, 3) pFliC 1+ GFP 1, 4)1 KB+, 5) pFliC 1+GFP 2, 6) pFliC 1 + GolBC, 7) pFliC 1 + GoIB1, 8) Ladder, 8) pFliC 1 + GoIB2, 9) pfliC 1 + fliC2 + GFP C, 10) pfliC 1+ fliC 2 + GFP 1, 11)pfliC1+ fliC 2 + GFP 2, 12) pfliC 1 + fliC2 + GFP 3
 
Jul-20Glycerol StockAndrewDH5α  
 Liquid CulturesBeiniTOP10  
 PCR Overlap ExtensionPhillip Andrew Thermopol Protocol:
Thermopol: 5 ul
dNTPs: 1 ul
Template: 10 ul
Vent Pol: 0.7 ul
MgSO4: 1 ul
H2O: 28.3 ul
Total: 33.3 ul

Using LP of fliC C1 and RP of respective part.

Program:1. Denaturation (Initial): 95 C for 5 mins 2. Denaturation: 98 C for 20 seconds 3. Annealing: 58 C for 20 seconds 4. Extension: 72 degrees, 45 seconds 5. Repeat step 2-4 for 15 cycles 6. Final extension 72 degrees, 5 minute 7. Cool down 4 degrees, one hour
 
 Motility Assay TestBeiniTOP 10 (Four colonies each)  
 Innoculation BL21 and XLI Blue using glycerol stocks with antibiotic  
 Gel ElectrophoresisPhillip, Faisal  Gel:
1) Blank, 2) Ladder, 3) pfliC1 + GFP 1, 4) pfliC1 + GFP 2, 5) pfliC1 + GoIB1, 6) pfliC1 + GoIB2, 7) Ladder, Rest is blank
 Gel Extraction Refer to Gel Extraction protocol  
Jul-21Motility Plate Results TOP 10 showed no motility. BL21 plate turned out fine  
 Motility Assay of BL21BeiniBL21  
 PCR of Gel ExtractionsBeinipfliC1 + GFP, pfliC1 + GoIBThermopol Protocol:
Thermopol: 5 ul
dNTPs: 1 ul
LP pfliC1: 1.5 ul
RP insert: 1.5 ul
DNA Template: 5 ul
Vent Pol: 0.7 ul
MgSO4: 1 ul
H2O: 28.3 ul
Total: 33.3 ul

Using LP of fliC C1 and RP of respective part.

Program:1. Denaturation (Initial): 95 C for 5 mins 2. Denaturation: 98 C for 20 seconds 3. Annealing: 60 C for 15 seconds 4. Extension: 72 degrees, 30 seconds 5. Repeat step 2-4 for 15 cycles 6. Final extension 72 degrees, 5 minute 7. Cool down 4 degrees, one hour
Gel:
1) Ladder 1KB+, 2) pFliC1 + GFP C, 3) pFliC 1+ GFP 1, 4) pFliC 1+GFP 2, 5) pFliC 1 + GolBC, 6) pFliC 1 + GoIB1, 7) pFliC 1 + GoIB 2, 8)Blank
Jul-22Motiliy Assay  XLI Blue  
 PCRAndrew Protocol not listed

Program:1. Denaturation (Initial): 95 C for 5 mins 2. Denaturation: 95 C for 20 seconds 3. Annealing: 60 C for 15 seconds 4. Extension: 72 degrees, 1 minute 5. Repeat step 2-4 for 15 cycles 6. Final extension 72 degrees, 5 minute 7. Cool down 4 degrees, one hour
 
 Glycerol StockFaisalTOP 10 Liquid Culture  
 Gel  Gel of round two PCR of gel extractionsGel:
1) Ladder 1KB+, 2) pFliC1 + GFP C, 3) pFliC 1+ GFP 1, 4) pFliC 1+GFP 2, 5) pFliC 1 + GolBC, 6) pFliC 1 + GoIB1, 7) pFliC 1 + GoIB 2, 8)Blank
 PCRBeini, AndrewpFliC1 + GFP (XS) + fliC2 (XS)Used Vent Protocol:

Program:1. Denaturation (Initial): 95 C for 5 mins 2. Denaturation: 95 C for 20 seconds 3. Annealing: 55 C for 30 seconds 4. Extension: 60 degrees, 1 minute 5. Repeat step 2-4 for 20 cycles 6. Final extension 72 degrees, 5 minute 7. Cool down 4 degrees, one hour
Gel:
1) Ladder, 2) pfliC1 + fliC2 +GFP C, 3)pfliC1 + fliC2 +GFP 1, 4)pfliC1 + fliC2 +GFP 2, 5)pfliC1 + fliC2 +GFP 2, 6) pfliC1 + SmtA C, 7) pfliC1 + SmtA 1, 8) pfliC1 + SmtA 2
 Motility Assay XLI Blue  
 PCRPhillippFLIC 1 +fliC2 + GFP (6 trials) and pFliC1 + fliC2 + GFP Control (3 rialsThermopol Protocol:
Thermopol: 5 ul
dNTPs: 1 ul
LP pfliC1: 1.5 ul
RP insert: 1.5 ul
DNA Template: 10 ul
Vent Pol: 0.7 ul
MgSO4: 1 ul
H2O: 28.3 ul
Total: 28.3 ul

Using LP of fliC C1 and RP of respective part.

Program:1. Denaturation (Initial): 95 C for 5 mins 2. Denaturation: 98 C for 20 seconds 3. Annealing: 58 C for 15 seconds 4. Extension: 72 degrees, 120 seconds 5. Repeat step 2-4 for 15 cycles 6. Final extension 72 degrees, 5 minute 7. Cool down 4 degrees, one hour

Monday July 16

To start the week off, Kevin went over some vital guidelines for lab work that we need to follow in order for us to keep organized and avoid wasting time/reagents. Then, since there were just the five of us, we all went into the lab to work on various things. Beini and Faisal did a FastDigest on 9 miniprep products to see if our ligations worked. They digested for 35 minutes instead of the usual 30, in hopes of better results. According to the gel, all the ligations failed. Andrew and Phillip performed a massive PCR of BioBrick parts we haven’t successfully PCR’d yet. Kevin did some reorganization of labwork, such as analyzing previous gels to check for digestion success.

Tuesday July 17th

This morning, Kevin and Andrew met with Dr. Smith to discuss gene sequences for cohesins and dockerins. Phillip and Andrew continued the gel analysis that Kevin started yesterday. Kevin, Faisal, and Beini worked on making primers for our next order. We planned to put in a rush order for only the primers that we would need to make a working GFP construct. In order for the primers to arrive tomorrow, we had to get our order in by 3 pm, so when everyone came back from lunch at 2 pm, we put all our heads together to grind out those primers. However, at 2:52 pm we ran into some unforeseen issues that required some consideration, so then we decided to chill out and just put an order in for 3 pm tomorrow. In the lab, we rehydrated a RBS BioBrick part and transformed it, liquid cultured some BL21 and XL1 Blue, and added a promoter to fliC1 using PCR.

Wednesday July 18th

Today, we put in our rush order for primers so that they hopefully arrive tomorrow. Kevin and Maggie met with a representative from KEDCO (Kingston Economic Development Corporation) to ask for advice on how to garner support for SynthetiQ from the local community (eg. educational institutions, dancers/choreographers). Later in the day, they also met with Dr. Allingham to discuss molecular modelling with a software called Coot, that we might use for modelling our chimeric flagellin. As for labwork, we made liquid cultures of the RBS from yesterday, made glycerol stocks of XL1 Blue and BL21, and digested fliC1 with SpeI to confirm the presence of that cut site. We also attempted a PCR overlap extension of pfliC1 with three different inserts (GFP, smtA, golB).

Thursday July 19th

Our rush order for primers was supposed to arrive by 5 pm, so we kept our eyes peeled for that the entire day, but alas they did not arrive by that time. For labwork, we re-ran yesterday’s PCR overlap extension and added more dNTPs and polymerase, because from the gel it looked like a lot of primers didn’t bind in the original PCR. We also tried PCR overlap extension using pfliC1 and X/S digests of fliC2 and GFP, with no primers. We attempted our first motility assay by stabbing a colony of DH5-alpha onto a soft agar plate. In the afternoon, we worked on primers for conversion of parts to BB-2 standard, and for parts we didn’t have primers for previously. Today was also Kevin’s birthday! Happy birthday Kevin.

Friday July 20th

For our motility assay of DH5-alpha, we got a nicely shaped circle ~1.1 cm. However, DH5-alpha is not supposed to express native flagella, so we will have to look further into this. For our next motility assay of TOP10, we decided to try one soft agar plate and one LB plate. We also divided the plates into quarters and tried four different levels of stabbing (i.e. from shallow to deep). For overlap PCR extension, we ran another round of PCR for pfliC1 + fliC2 + GFP, this time just adding a bit more polymerase and not dNTPs (because they probably didn’t get used up). Then we ran a gel of all the PCR overlap extensions we have done so far. In the afternoon, Kevin showed us how to do a gel extraction, and cut out bands of fliC1 + GFP and fliC1 + GolB from the aforementioned gel. For the rest of the day, Phillip did some filming for our project overview video as we took turns speaking for the camcorder.