Week from Semptember 7th, to September 13th, 2009

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September, 7th

In the morning we have prepared colture of A2 to stock in glycerol and we have prepared LB medium.

In the afternoon we have incubated in adequate medium:

• B9 (10 ml LB+Amp)
• B10 (10 ml LB+Amp)
• T9002 (10 ml LB + Amp)

for a qualitative test of ethanol production,

• A9pg (5 ml LB + Amp)
• A16 (5 ml LB + Amp)
• A8 (5 ml LB + Amp)
• A2 (5 ml LB + Amp)
• RBS33 (5 ml LB + Amp)

to perform an aTc induction test,

• A15 (25 ml LB + Amp)
• pSB3K3 (25 ml LB + Kan)
• A14-1 L (5 ml LB + Amp)

to miniprep tomorrow.

These coltures have been incubated at 37°C, 220 rpm overnight. Tomorrow, we will miniprep A14-1 L and midiprep A15 and 3K3 (pSB3K3). We perform midiprep on pSB3K3 because it is a low copy number plasmid and on A15 because it is a lysis cassette, so for leackage only "few" cells survive.

Glycerol stocks of A2-3 have been stored at -80°C, named A2-3 and A2-3 bis.The previous stock has been thrown away.

Preparation of a qualitative test to assay the presence of ethanol

We have also induced coltures B9 and B10 incubated yesterday morning at 37°C 220 rpm in flasks and grown overnight. In the morning the coltures have been diluited to reach the desired OD (0.03), then re-incubated for 4 hours and induced with 3OC6-HSL 10nM. The induced coltures have been incubated at 18.00 o'clock and will stay overnight. Flasks have been ermetically closed, to allow fermentation. This qualitative test is performed to assay the presence oh ethanol.

Tomorrow we will use the "Ethanol assay kit" to test the presence of ethanol!!

September, 8th

A first test with ethanol assay kit has been made using coltures B9 and B10 induced the evening before and using as blank the reaction mix of Kit without ethanol. In the evening, we have also made a measure of blank made by LB + mix. The test have been performed on the supernatant of the coltures, obtained by centrifugation for 20 minutes 13000rpm.

• A14 L-1 have been miniprepped, with a yield of 33ng/ul
• pSB3K3 and A15 have been midi-prepped and tomorrow will be digested E-P and ligated

aTc induction test:

• one of the coltures incubated the day befoore has been "accidentally" centrifugated, and has shown no growth in the next 3 hours, so the coltures have been re-inoculated, as the day before:
• A9pg (5 ml LB + Amp)
• A16 (5 ml LB + Amp)
• A8 (5 ml LB + Amp)
• A2 (5 ml LB + Amp)
• RBS33 (5 ml LB + Amp)

to perform a new aTc induction test, tomorrow.

September, 9th

Today we made aTc induction test:

• coltures have been diluted 1:100 in the morning (50 ul of colture incubated overnight at 37° 220 rpm in 5 ml of new LB+Amp fresh medium) and incubated in the same conditions for further 4 hours
• in the afternoon they have been diluted again to start from the desired O.D. of 0,02 using the usual phormula.
• Implementation of induction test with aTc

• Ligation of pSB3K3 with A15: digestion of both the BioBrick E-P, gel extraction and ligation overnight.
• B9 B10 and F620 have been inoculated from glycerol stocks to be used tomorrow for thanol assay test.

We have also received the resultes of A17 sequencing. In the afternoon we have made a qualitative ethanol assay test: coltures induced yesterday with 3OC6HSL 10nM have been centrifugate and supernatant has been diluted:

• 1:400
• 1:100
• 1:10

The measure was not significant for the Ethanol Assay Kit doesn't work.

September, 10th

The ligation of A15 and pSB3K3, named A19, has been tranformed in E. coli TOP10. The coltures B9, B10, F2620 have been diluted 1:100 and induced with 3OC6HSL 100nM to be tested, with glucose 10% added.

Test aTc with good results in LB

September, 11st

The lab has been closed for techinal problems, so we have just performed test at TECAN

Ethanol test: ethanol assay kit doesn't work!!! We've tested coltures induced woth 3OC6HSL 10nM and with 10% glucose added. The measure has been made on the whole cultures, not centrifugated, for we couldn't have access to the lab. No result has been obtained for the enzyme of ethanol assay kit doesn't work!

At 16.00, meeting of our team with representatives of Costa Foundation.

September, 12nd

September, 13rd

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