Team:UNIPV-Pavia/Calendar/July/settimana1

JULY: WEEK 1

June, 28th

Inoculum from glycerol stock for I7-1, I7-2, I8-1, I8-2, I9-1, I9-2, I10-1 and I10-2 in 5ml LB+Amp. Cultures were gown ON at 37°C 220rpm.

Inoculum of PhaP-1 and PhaP-2 in 5ml LB+Kan and ON growth at 37°C 220rpm.

We received strains and plasmid from Yale University

June, 29th

All cultures were grown. From I7-1, I7-2, I8-1, I8-2, I9-1, I9-2, I10-1 and I10-2 5ul were aliquoted and diluted in 500ul LB+Amp for a preliminary TECAN test. Cultures were MiniPrepped with the following quantifications (NanoDrop):

All cultures, except PhaP-1 and PhaP-2 were digested. Digestion of:

Digestion was performed at 37°C for 3 hours. Digestions were gel run. For I9 and I10 clones all colonie were correct, while for I7 and I8 extra bands could be observed. Since preliminar results of TECAN test didn't provide encouraging results, we decided to select other 3 colonies both for I7 and I8 and to repeat the screening, while I9 and I10 were correct at this screening, so we chose I9-1 and I10-1 as definitive I9 and I10 parts. I9 and I10 will be further screened next week.

TECAN TEST

Screening of I7, I8, I9, I10

PhaP-1 and PhaP-2 were prepared or sequencing: 13,8 ul (2x quantity) of PhaP-1 and 14,7ul (1x quantity) oh PhaP-2 were dryed at 65°C and setn to BMR genomics for sequencing service.

All strains received from Yale Univesity were on blotting paper disks, that were placed on an LB agar plates (with/without antiobiotic, see growth conditions), resuspended wih 80ul LB and then streaked. After plate streaking, disks were trasnferred in falcon containing liquid LB.

• BT340 was streaked on LB+Amp (50ng/ml) agar plates and from here on LB+Amp (100ng/ml) agar plates. The paper disk was then transferred in liquid LB+Amp (100ng/ml).
• BW5328 was streaked on LB+Amp (50ng/ml) agar plates and from here on LB+Amp (100ng/ml) agar plates. The paper disk was then transferred in liquid LB+Amp (100ng/ml).
• MG1655 was streaked on LB agar plates. The paper disk was then transferred in liquid LB.
• MC1061 was streaked on LB agar plates. The paper disk was then transferred in liquid LB.
• BW25142 was streaked on LB agar plates. The paper disk was then transferred in liquid LB.
• BW25141 was streaked on LB agar plates. The paper disk was then transferred in liquid LB.

METTERE CONDIZIONI DI CRESCITA (temperatura e tempo) e risultati (chi è cresciuto e chi no!)

June, 30th

Inoculum of cultures for a TECAN test. From glycerol stocks: I7-1, I7-2, I8-1, I8-2 (all tese 4 colonies were not correct! we want to see if any fluorescence is observed in order to understamd if there is any unexpected GFP production), I9-1, I9-2, I10-1, I10-2. For these cultures 8ul of glycerol stock were inoculated in 2 ml Lb+Amp. Other three colonies were peaked from I7 and I8 plates.

All cultures were incubated ON, 37°C 220 rpm.

July, 1st

First TECAN test.

All cultures incubated yesterday night were diluted 1:100 (10ul in 1ml fresh LB+Amp) and let grown for further 3 hours at 37°C 220 rpm. Also I7-3,4 and 5 and I8-3, 4 and 5 were prepared for TECAN test (10ul were diluted in 1ml LB+Amp). After dilution, static OD was measured for these cultures and a proper dilution was perfeormed, in order to start from the desired OD of 0,02, according to the phormula:

After dilution, 3 aliquotes each of 200ul were trasferred in a 96-wells plate and incubated in TECAN multi-well plate reader. Cultures were mantained at 37°C and a kynetic cycle was performed:

• Duration: 20h
• Kynetic cycle: 5 minutes
• Every 5 minutes:
  • 15 s shaking (3mm, linear)
  • 10 s wait
  • OD600 measurement
  • GFP measurement

I7-3, I7-4, I7-5, I8-3, I8-4 and I8-5 cultures were prepared in 5ml LB+Amp, so the remaining cultures not used for TECAN test were pelletted at 4000 rpm for 10 minutes, supernatant was discarded and pelletes were stored at 4°C for tomorrow MiniPrep of cultures that provided promising results at TECAN test.

Of the plates incubated yesterday, a colony was peaked and grown in 1ml LB (+ antibiotic) for 6 hours at X°C 220 rpm:

Other parts were received from Anderson Lab. in stab form.

Plates were incubated at 37°C ON.

July, 2nd

TECAN test provided encouraging results, showing that I7-3, I7-5, I8-4 and I8-5 produced GFP. Also I9-1, I9-2, I10-1 and I10-2 confirmed the preliminar results of the precious test, in fact GFP production was observe. At a preliminary analysis, it seems that GFP production is correlated to the srength of the promoter regulating luxI production!! :) Other culture (I7-1, I7-2, I7-4, I8-1, I8-2, I8-3) did not produce GFP, se they were thrown away. A furhter screening were performed on I7-3, I7-5, I8-4, I8-5, I9-1 and I10-1.

For this reason, MiniPro was performed for the following colonies, with the following quantiications:

I7-3, I7-5, I8-4 and I8-5 were digested with EcorI and PstI. All colonies were digested with NheI-PstI (2 NheI sites are present in the BBa_J23xx promoter, so this digestion shows if the promoter has been ligated.)

Digestion of:

Digestions were incubated at 37°C for 3 hours and gel run.

Screening of new colonies of I7 and I8. New screening for I9, I10

This time all the clones are OK :)

We choose the following colonies: