Team:Cambridge/LabBook/Week4

Monday

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.

Tuesday

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:

OD₆₀₀ measurements:

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