Team:Cambridge/LabBook/Week3

27.07.10

1. Experiment: Streaking out of bacterial cultures (Peter & Anja)

On LB agar plates:

• TOP10
• MG1655
• W3110 hns 93-1
• BW25113

On LB agar + kan plates:

• BW25113 Δhns::kan
• BW25113 ΔtraA::kan

Incubated at 37°C overnight

28.07.10

all strains grew with individual colonies.

2. Experiment: Set up overnight culture of TOP10 (Emily & Anja)

Inoculated single bacterial colony (ATP10) in 12ml LB, incubated at 37°C with shaking (180 rpm) overnight.

29.07.10

Inoculated 400ml LB with 5ml TOP10 overnight culture, incubate at 37°C with shaking (180rpm) put on at 11:40am

OD₆₀₀ measurements:

Time	OD600 (1)	OD600 (2)
12:20pm	-0.026	-0.032
2:30pm	0.437	0.530

3. Preparation: CCMB80 Buffer (Volume500ml)

Arrows label dilutions to the indicated concentration

• KOAC 1M ---> 10mM 5ml
• CaCl₂.2H₂0 1M ---> 80mM 40ml
• MnCl₂.4H₂0 1M ---> 20mM 10ml
• MnCl₂.6H₂0 1M ---> 10mM 5ml
• glycerol --->10%
• sterile H₂0 390ml
• prepare in flow hood
• sterile filter
• store at 4°C

4. Experiment: Set up overnight cultures of TOP10 (Will & Anja)

Inoculated single bacterial (TOP10) colony in 5ml SOB, incubated at rtp with shaking overnight

30.07.10

5.Experiment: Preparing chemicall competent cells (Emily, Bill & Anja)

(followed protocol for 'TOP10 chemically competent cells' from OpenWetWare)

Inoculated 800ml SOB with 3ml of TOP10 overnight culture (grown from single colony), incubated at 37°C with shaking (180rpm)

Put on at 9.45am

OD₆₀₀ measurements:

Time	OD600 (teaching lab)	OD600 (Jim's lab)
10:50am	0.008	-0.002
11:30am	0.013	0.000
12:00pm	0.023	0.024
12:35pm	0.032	0.034
01:00pm	0.040	0.046
01:32pm	0.052	0.063
02:00pm	0.073	0.090
02:35pm	0.106	0.140
03:00pm	0.137	0.174
03:25pm	0.179	0.230
03:38pm	0.203	0.256
03:55pm	0.225	0.313

Cooled cells and CCMB80 buffer in ice bath for around 20min. Aliquoted 600ml culture in 12x50ml centrifuge tubes (pointed bottom). Centrifuged at 3000g at 4°C for 10min. Discarded supernatatent. Gently resuspended cell pellet in 20ml ice cold CCMB80 buffer per tube. Incubated on ice for 20min. Centrifuget at 3000g at 4°C for 10min. Pooled Pairs of tubes together. 12 tubes ---> 6tubes. Discarded supernatant. Resuspended pellet in 5ml ice for 20min. Aliquoted 150x200μl into Eppendorf tubes. Stored at -80°C.

31.07.10 & 01.08.10

6. Experiment: Measuring competency of TOP10 (Fernan)

TOP10 coopmetent cells (cc) taken out of -80°C freezer and put on ice. Check for thawing ater 5minutes (leave on ice). Cut 1ml pipette tips with scissors sterilised in ethanol and flamed with a Bunsen burner. Transfer ~50μl of TOP10 cc into 1.5ml Eppendorf tube with cut pipette tip. (will have to judge ~50μl by eye). Added 1μl of pUC19 (standard control plasmid) at 10^-5μg/μl. Held on ice for 30min. Heat shockede for 60s at 42°C (waterbath). Put on ice for ~2min. Added 250μl pre-warmed (in 37°C incubator) SOC. Incubated at 37°C for 1h with rotation (for this purpose stick Eppendorf tubes in 12ml falcon tubes and put tape over the top). Plated 20μl on LB agar paltes with Amp (with blue shaped spreader). Grow colonies overnight at 37°C.

• Yielded ~150 cells/plate
• transformation efficiency (no. of transformed cells (colonies) generated by 1μg of plasmid DNA)
• volume of cells * colonies on a plate / mass of DNA transformed
• 15 * 150 * 10⁵ = 2.25x10⁸/ μgDNA
• volume=50μl cells + 1μl plasmid + 250μl SOC, 301μl of which 2Oμl were plated, giving dilution factor of 15
• mass of DNA is 10^-5 μg

02.08.10

7. Experiment: Transformation of TOP10 cc (Ben, Emily, Bill, Hannah, Will & Anja)

Top10 cc taken out of -80°C freezer and tawed on ice. 1ml pipette tip cut with scissors sterilised with ethanol and flamed. Using cut pipette tip ~50μl of TOP10cc were transferred to 1.5ml Eppendorf tubes (3x)

1. 2μl of resuspended (in 10μl deionised water) BBa_J13002 (plasmiid with Tet^R represed PoPs/RIPs generator) was added.
2. 2μl of resuspended (in 10μl deionised water) BBa_I712019 (plasmid with firefly luciferase) was added
3. 1.5μl of pHK724 (plasmid containing lux4 gene) was addded

Ce]]s were held on ice for 30min. Heat shocked for 60s at 42°C (waterbath). As a control TOP10cc that had not been transformed with anything (no plasmid DNA added) were subjected to same treatment (as cells to be transformed). Put on ice for ~2min. Added 250μl pre-wardmed (in 37°C) SOC. Incubated at 37°C for Ph with rotation (Eppendorf tubes---> 12ml falcon tubes, tape over top). Plated 50micorl on pre-warmed (in 37°C incubator) LB agar plates with Amp (with blue L-shaped spreader). Grow colonies overnight at 37°2.

8.Experiment: Set up overnight culture of E.coli/pHK555 (Will & Anja)

Inoculated single bacterial colony (E.coli/pHK555) in 5ml LB in a 12ml falcon tube. Incubated at 37°C with rotation overnight. (Since it was difficult to see the colony sent from Jim Slock, we picked twice from where we anticipated colony to be and set up 2x5ml LB overnight cultures)

9.Experiment: Set up overnight cultures of W3110 hns 93-1, BW25113 Δhns::kan, W3110 hns-205::Tn10 Tet^R, GM230 hns-205::Tn10 Tet^R (Ben & Anja)

Inoculated single bacterial colonies in 5ml SOB (i.e. 4 different cultures), incubated at RT with shaking overnight.

03.08.10

10. Experiment: Preparing chemically competent cells (W3110 hns 93-1, BW25113 Δhns::kan,W3110 hns-205::Tn10 Tet^R, GM230 hns-205::Tn10 Tet^R (Ben, Will, Paul, Bill, Emily, Hannah & Anja)

(followed protocol for 'TOP10 chemically competent cells cells' from OpenWetWare) Inoculated 0.5ml of the 4 bacterial strains (overnight cultures) in 85ml SOB each. Incubated at 37°C with shaking (180rpm). put on at 11:55am

Bacterial Strains:

ID	Type
(1)	GM230 hns-205::Tn10 TetR
(2)	BW25113 Δhns::kan
(3)	W3110 hns 93-1
(4)	W3110 hns-205::Tn10 TetR

OD₆₀₀ measurements:

Time	OD600 (1)	OD600 (2)	OD600 (3)	OD600 (4)
1:00pm	0.019	-0.008	-0.020	0.049
2:00pm	0.034	0.027	0.001	0.124
3:00pm	0.059	0.114	0.049	0.268
3:25pm	0.092	0.235	0.116	-
3:40pm	-	0.297	-	-
4:00pm	0.159	-	0.267	-
4:25pm	0.203	-	-	-
4:40pm	0.256	-	-	-

Cooled cells and (newly prepared) CCMB80 Buffer in ice bath for ~20 minutes. (4) was on ice for over an hour. (2) was on ice for ~40 minutes. (3) was on ice for ~40 minutes.

2 x 30-35ml of each of the four 85ml cultures were poured into 50ml falcon tubes. Centrifuged at 3000g at 4°C for 10 min. Discarded supernatant. Resuspended cell pellet in 20ml tube CCMB80 buffer (ice cold). On ice for 20 min. Centrifuged at 3000g at 4°C for 10 min. Discarded supernatant. Resuspended cell pellet in 5ml tube CCMB80 buffer. Incubated on ice for 20 min. Aliquoted 200μl portions into Eppendorf tubes colour-coded according to the above listed colour labels. Stored at -80°C

11. Experiment: Streaking out of bacterial cultures (E. coli/pHK555) (Paul & Anja)

On LB agar plates with Chloranphenicol: E. coli/pHK555 (from both overnight cultures) on two individual plates incubated at 37°C overnight.

12. Experiment: Set up overnight cultures (TOP10 with BBa-J13002, TOP10 with BBa_I712019) (Ben & Anja)

Results from transformations on 02/08/10

• Colonies grew for TOP10 cells transformed with BBa_J13002 (promoter + rbs) and those transformed with BBa_I712019 (firefly luciferase) :-)
• no cells grew for untransformed TOP10 cells plated on LB agar + Amp plates (neg control)
• small (but plenty) colonies grew for TOP10 cells transformed with pHK724, these were left in the incubator for another 24h, and then put at 4°C

Inoculated single bacterial (TOP10 with BBa-J13002 and TOP10 with BBa_I712019 individually) colony in 5ml LB, incubated at 37°C with shaking (180rpm) overnight