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From 2013.igem.org

Contents 1 Morning 2 Afternoon 3 Transformation of Competant Cells 4 (Next day, results of transformation)

Morning

• Made 1 litre of soy trypticase agar using :

5 g NaCl

15 g Tryptone

15 g Agar

5 g Soytone

Tryptone, NaCl and soytone were added into a mixer with a magnetic stirrer. (was a yellow colour).

3 g of agar were added to two 200ml durans. The Tryptone mix was then added to the durans, filling it up to 200ml.

Mixture was yellow, not colourless - the experiment was not successful. Durans put in the autoclave in the evening to let the agar set overnight.

Afternoon

Transformation of Competant Cells

(This protocol will be used throughout our project, however the standard protocol from iGEM was modified slightly)

1. Competent cells were thawed on ice.

2. 50 µl of thawed competent cells was added into a pre chilled 2 ml tube, and another 50 µl was added to a different 2ml tube which was labelled 'control'.

3. 2 µl of the resuspended DNA was added to the 2 ml tube. This was pipetted up and down gently a few times. Competent cells were kept on ice.

4. 2 µl of the RFP Control was added to the control transformation.

5. Tubes were closed and cells were incubated on ice for 30 minutes.

6. The cells were heat shocked by immersion in a pre-heated water bath at 42°C for 60 seconds.

7. Cells were incubated on ice for 5 minutes.

8. SOC media was made by the addition of 500 µl of glucose to SOB. 500 µl of SOC media was added to each transformation.

9. Cells were incubated at 37°C for 1.5 hours while the tubes were shaking.

10. 2 petri dishes were labelled with the part number/control, plasmid backbone and antibiotic resistance.

11. Pipette 100ul of the cells onto one of the plates, and spread using a sterile spread stick.

12. Centrifuge the remaining cells for 2 mins at 13,000 rpm. Pour off the supernatant and resuspend the pellet in 100ul of fresh SOC. Plate this out.

13. Incubate the plates at 37°C for 12-14 hours overnight (make sure they're upside-down!)

The team divided into 3 groups...

- One transformed BBa_I13522, a GFP on pSB1C3

- One transformed BBa_J04450, a RFP on pSB1C3

- One transformed BBa_K322124, which codes for cph8 red light sensor and we need to send off for sequencing.

Overall, each team made 4 plates. One with the gene of interest and a low concentration of bacteria, one with the gene of interest and a high concentration of bacteria, and two with the RFP control at the two different concentrations of bacteria.

(Next day, results of transformation)

cph8 team

cph8 low density plate - no colonies

cph8 high density plate - 7 colonies

RFP control low density plate - 98 colonies

RFP control high density plate - 376 colonies

GFP team

GFP low density plate - 35 colonies

GFP high density plate - 178 colonies

RFP control low density plate - 104 colonies

RFP control high density plate - 684 colonies