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From 2010.igem.org

Notebook	Building Parts	Testing Parts	Assembly Method	Competent Cells	Plates	Kit Manual	Software

Plates

Sintered Plastic

22-06-10

Creation of a reusable surface on which to grow bacteria.

Supplier: SPC Technologies Ltd. Sintered Plastic Samples

1) 3.0mm Ultra-Fine PE Sheet - mean pore size 14µm

2) 6.0mm Fine Grade PE Sheet - mean pore size 30µm

3) 4.5mm Medium Grade PE Sheet - mean pore size 88µm

• according to materials sheet, the ultra-fine sheet is a bacterial barrier

Procedure:

1) cut each 100mm x 100mm sample with an exacto-knife into into disks approximately 100mm in diameter and inserted into petri dishes

2) added 10ml of LB and 40µl of chloranphenicol to each plate

3) incubated the plates overnight at 37C

Observations:

- 6.0mm fine grade sheet was to thick to cut with an exacto knife and so was not used

- both sheets did not absorb the LB immediately; the LB beaded on the surface

23-06-10

Observations:

- 7mL LB left from 3.0mm ultra-fine sheet (3mL absorbed)

- 8mL LB left from 4.5mm medium-grade (2mL absorbed)

- 3.0mm ultra-fine PE sheet with mean pore size 14µm is more absorptive

Procedure:

1) transferred both plastic disks into new, separate petri dishes

2) streaked plastic sheets with RFP-containing colonies

3) incubated in 37C overnight

24-06-10

- no growth after incubation

- plastic very dry

- plastic sheets deemed too hydrophobic to retain aqueous LB

Procedure:

1) rubbed small amount of ethanol onto each plastic disk

2) followed by rubbing in milli-Q water to decrease the surface tension

Observations:

- absorbency visibly increased

1) plastic was soaked in 95% ethanol and then put into the dessicator to dry out so that the plates are now reusable and sterile

Researched into hydrophilic sintered plastic. Possible, but too expensive to consider for the purpose of our project

Jello and Gelatin

26-07-10

<p>Creation of a reusable, inexpensive, surface to grow bacteria on

A) Jell-O, no sugar added (sweetened with aspartane)

ingredients - 125mg sodium per 2.5 and 1g protein per 2.5g

Procedure:

1) stirred 2 cups boiling water into 10g of jell-o powder

2) stirred until dissolved and poured into petri dish

3) chilled for 2 hours

B) Knox Gelatine

ingredients - 1g protein per 1.8g

Procedure:

1) stirred in 1/2 cup (175ml) of room temperature Milli-Q into one pouch (15ml) of Gelatine

2) stirred in 1/2 cup of hot milli-Q (microwaved for 1 min.)

3) poured solution into petri dishes

4) put petri dishes in -4C until set (took approximately 2 hours)

Spread BBa_k274003 dark green strain onto two jell-o and two gelatine plates and left one of each of the plates bacteria free for controls. Incubated in 37C overnight.

27-07-10

Both the jell-o plates and gelatine plates had liquified and no growth was present. Melting point for gelatine is 35C.

L.B.O.

24-06-10

Creation of "L.B.O.": a deodorant stick of LB agar

All of the following steps were performed in a safety cabinet under sterile conditions.

1) disassembled a Degree deodorant stick and soaked in ethanol

2) removed the raising platform and covered it with Parafilm

3) coated the insides of the tube with mineral oil

5) removed platform and poured LB agar into the base of the stick, waited until it solidified

6) the Parafilmed platform was put back into the stick and lowered maximally

7) LB agar was poured on top of the platform, until the tube was full, waited until it solidifed

8) The top of the LB agar was sliced off with an ethanol-sterilized knife

9) 400ul of transformed competent cells were plated on the stick as if it was a standard LB agar plate

10) L.B.O. stick was incubated overnight at 37C

Observations:

- although the knife cut through the LB agar easily, it was difficult to produce a completely flat surface

- solidified LB agar was effectively raised and lowered using the dial

- after incubation, a bacterial lawn was observed and a distinct E.coli smell was present

29-06-10

1) Followed procedure from June 26, 2010 to put new LB agar into the L.B.O. tube

2) Approximately 1cm was sliced off the top of the new LB agar with an ethanol-sterilized razor blade

3) L.B.O. stick was incubated overnight at 37C

Observations:

- no growth observed, tube must have been sterile

30-06-10

1) Streaked green colony from a plate of Cambridge parts

2) L.B.O. stick was incubated overnight at 37C

Observations:

- green streak and individual green colonies observed, along with a distinct E.coli smell

05-07-10

1) Approximately 1cm was sliced off the top of the used LB agar with an ethanol-sterilized razor blade

2) Streaked red, RFP-containing colony onto the LB agar surface.

3) L.B.O. stick was incubated overnight at 37C

Observations:

- only a red streak and individual red colonies observed, along with a distinct E.coli smell

- no remnants of green colonies visible