Team:UNIPV-Pavia/Calendar/June/settimana3

June, 7th

These BioBrick were resuspended in 15ul ddH20:

• BBa_E2050 (mOrange, 744bp) 2010 Kit Plate 2, well 13N in pSB2K3
• BBa_K165018 (ADH1 terminator, 253 bp) 2010 Kit Plate 3, well 2M, J63009 (Amp)
• BBa_K165037 (tef2 promoter, 403bp) 2010 Kit Plate 3, well 22O, pSB1AK3
• BBa_P1004 (Chloramphenicol resistence cassette, 769 bp) 2009 Kit plate 1, well 7B, pSB1A1
• BBa_J61001 (R6K origin, 406bp) 2010 Kit plate 1, well 240, pSB1A2
• BBa_K081008 (RBS-luxI, 664bp) 2010 Kit plate 2, well 10L, pSB1A2
• BBa_K125500 (GFP fusion brick, 718bp) 2010 Kit plate 3, well 2P, pSB1A2

1,5 ul of each culture was transformed in 100 ul of home-made competent E. coli DH5alpha.

Tranformants were all plated on LB agar plates added with Ampicillin, except for BBa_E2050 (Kanamycin).

500 ml LB+Amp was prepared and 21 LB agar plates + Amp were prepared (500 ml).

June, 8th

All plates grown overnight at 37°C showed colonies! In particular, BBa_E2050 (grown on LB+Kan), BBa_K125500 (grown on LB+Amp), BBa_K081008 (grown on LB+Amp) and BBa_K165018 (grown on LB+Amp) showed big colonies, well separated on the plate. BBa_P1004 (grown on LB+Amp) and BBa_J61001 (grown on LB+Amp) showed many colonies, with big colonies surrounded by small colonies. BBa_K165037 (grown on LB+Amp) showed only 11 big colonies.

A colony was picked for each plate and inoculated in 1ml LB+antibiotic.

BBa_E2050 1ml LB+Kan BBa_K165037 1ml LB+Amp BBa_P1004 1ml LB+Amp BBa_K125500 1ml LB+Amp BBa_K081008 1ml LB+Amp BBa_J61001 1ml LB+Amp BBa_K165018 1ml LB+Amp

Sara and Nicolò at work

BBa_K165037 in pSB1AK3 plasmid was inocultaed both in 1ml LB+Amp and in 1ml LB+Kan for a phenotipic assay.

Cultures were grown for 8 hours at 37°C 220 rpm.

After this time, cultures were all grown and in saturation phase. BBa_K165037 in LB+Kan was also grown, so that this phenotipic assay was positive.

Glycerol stocks were prepared for each culture (250ul saturated culture + 750 ul glycerol 80%) and stored at -80°C.

The left culture was re-filled to 5ml with LB+antibiotic and grown overnight at 37°C 200 rpm to be miniprepped tomorrow.

June, 9th

Ligation of:

• I0: BBa_K125500 (E-S)+BBa_B0015 (E-X)
• I1: BBa_E2050 (E-S)+BBa_K165018 (E-X)
• I2: BBa_P1004 (E-S)+BBa_B0015 (E-X)
• I3: BBa_K081008 (E-S)+BBa_B0015 (E-X)

BBa_B0015 was accidentally not inoculated yesterday... :( Luckily, we had a DNA stock for this part stored at -20°C, so we used this stock for ligations.

Cultures grown overnight at 37°C 220 rpm were all saturated. BioBricks were extracted with MiniPrep kit. Surprisingly, after the first centrifugation, we observed that BBa_E2050 pellet was orange!! This is curious, because this BioBrick expresses mOrange, but it lacks of promoters. Probably, this spurious transcription is due to the promoter of Kanamycin resistance, that is oriented in the same direction.

BBa_E2050 orange pellet

After MiniPrep, purified DNA was quantified with NanoDrop. BBa_E2050 139,2 ng/ul BBa_K165037 181,5 ng/ul BBa_P1004 132,8 ng/ul BBa_K125500 124,3 ng/ul BBa_K081008 138,3 ng/ul BBa_J61001 122,2 ng/ul BBa_K165018 166,3 ng/ul

Digestion of:

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

Gel run/cut of cultures

Ligation of I0, I1, I2 and I3 was performed at 16°C overnight.

June, 10th

I0, I1, I2 and I3 were tranformed in home made compotent E. coli DH5-alpha and plated on LB+Amp agar plates.

Plates with transformed E.coli were incubated overnight at 37°C 220 rpm.

The four plates were named:

• iGEM 2010 10/06/2010 DH5-alpha I0 LB+Amp
• iGEM 2010 10/06/2010 DH5-alpha I1 LB+Amp
• iGEM 2010 10/06/2010 DH5-alpha I2 LB+Amp
• iGEM 2010 10/06/2010 DH5-alpha I3 LB+Amp

and stored at +4°C.

June, 11th

All four plates grown overnight showed colonies!! Two colonies for each plate were picked and incubated in 1ml LB+Amp. For I2, a phenotipic test was performed: the same two colonies were inoculated both in LB+Amp and LB+Cm. So, 10 cultures were incubated at 37°C 220 rpm for 8 hours:

After over-day growth, we observed that the phenotipic test for I2 was positive for both colonies: I2-1 and I2-2 were grown both in LB+Amp and LB+Cm.

8 glycerol stocks were prepared and stored at -80°C. Next week a screening will be performed to select positive colonies.