Team:UNIPV-Pavia/Calendar/June

JUNE

June, 7th

These BioBrick were resuspended in 15ul ddH20:

• <partinfo>BBa_E2050</partinfo> (mOrange, 744bp) 2010 Kit Plate 2, well 13N in pSB2K3
• <partinfo>BBa_K165018</partinfo> (ADH1 terminator, 253 bp) 2010 Kit Plate 3, well 2M, J63009 (Amp)
• <partinfo>BBa_K165037</partinfo> (tef2 promoter, 403bp) 2010 Kit Plate 3, well 22O, pSB1AK3
• <partinfo>BBa_P1004</partinfo> (Chloramphenicol resistence cassette, 769 bp) 2009 Kit plate 1, well 7B, pSB1A1
• <partinfo>BBa_J61001</partinfo> (R6K origin, 406bp) 2010 Kit plate 1, well 240, pSB1A2
• <partinfo>BBa_K081008</partinfo> (RBS-luxI, 664bp) 2010 Kit plate 2, well 10L, pSB1A2
• <partinfo>BBa_K125500</partinfo> (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 <partinfo>BBa_E2050</partinfo> (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 show colonies! In particular, <partinfo>BBa_E2050</partinfo> (grown on LB+Kan), <partinfo>BBa_K125500</partinfo> (grown on LB+Amp), <partinfo>BBa_K081008</partinfo> (grown on LB+Amp) and <partinfo>BBa_K165018</partinfo> (grown on LB+Amp) showed big colonies, well separated on the plate. <partinfo>BBa_P1004</partinfo> (grown on LB+Amp) and <partinfo>BBa_J61001</partinfo> (grown on LB+Amp) showed many colonies, with big colonies surrounded by small colonies. <partinfo>BBa_K165037</partinfo> (grown on LB+Amp) showed only 11 big colonies.

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

<partinfo>BBa_E2050</partinfo> 1ml LB+Kan <partinfo>BBa_K165037</partinfo> 1ml LB+Amp <partinfo>BBa_P1004</partinfo> 1ml LB+Amp <partinfo>BBa_K125500</partinfo> 1ml LB+Amp <partinfo>BBa_K081008</partinfo> 1ml LB+Amp <partinfo>BBa_J61001</partinfo> 1ml LB+Amp <partinfo>BBa_K165018</partinfo> 1ml LB+Amp

Sara and Nicolò at work

<partinfo>BBa_K165037</partinfo> 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. <partinfo>BBa_K165037</partinfo> 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: <partinfo>BBa_K125500</partinfo> (E-S)+<partinfo>BBa_B0015</partinfo> (E-X)
• I1: <partinfo>BBa_E2050</partinfo> (E-S)+<partinfo>BBa_K165018</partinfo> (E-X)
• I2: <partinfo>BBa_P1004</partinfo> (E-S)+<partinfo>BBa_B0015</partinfo> (E-X)
• I3: <partinfo>BBa_K081008</partinfo> (E-S)+<partinfo>BBa_B0015</partinfo> (E-X)

<partinfo>BBa_B0015</partinfo> 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 <partinfo>BBa_E2050</partinfo> 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. <partinfo>BBa_E2050</partinfo> 139,2 ng/ul <partinfo>BBa_K165037</partinfo> 181,5 ng/ul <partinfo>BBa_P1004</partinfo> 132,8 ng/ul <partinfo>BBa_K125500</partinfo> 124,3 ng/ul <partinfo>BBa_K081008</partinfo> 138,3 ng/ul <partinfo>BBa_J61001</partinfo> 122,2 ng/ul <partinfo>BBa_K165018</partinfo> 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 are stored at +4°C.

June, 11th

All four plates grown overnight showed colonies!! Two colonies for each plate were peaked 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.

June, 14th

Inoculum in 5ml LB+Amp from glycerol stock for:

• I0-1
• I0-2
• I1-1
• I1-2
• I2-1 (phenotipic screening ok, grown both on LB+Amp and LB+Cm)
• I3-1
• I3-2

Cultures were grown overnight at 37°C 220 rpm.

June, 15th

Cultures grown overnight at 37°C 220 rpm were all saturated. BioBricks were extracted with MiniPrep kit.

After MiniPrep, purified DNA was quantified with NanoDrop.

Digestion of:

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

Two gels were prepared, as shown in figure.

Gel results show that:

• I0-1 is negative, while I0-2 is positive: we choose I0-2, from now on named I0, for sequencing and for gel extraction of Insert.
• Both I1-1 and I1-2 are positive. We choose I1-2, from now on named I1, for sequencing and gel-extraction because its DNA concentration is better.
• I2-1, from now on I2, is positive so it will be used for sequencing and gel-extraction.
• I3-2 is negative, while I3-1, from now on I3, is used for sequencing and gel-extraction.

So I0-2, I1-2, I2-1 and I3-1 samples are prepared for sequencing.

Ligation of:

• I4: <partinfo>BBa_K165037</partinfo> (S-P) + I1 (X-P)
• I5: I2 (E-S) + <partinfo>BBa_J61001</partinfo> (E-X)
• I6: I3 (E-S) + <partinfo>BBa_T9002</partinfo> (E-X)

Ligation of I4, I5 and I6 was performed at 16°C overnight.

June, 16th

Ligations I4, I5 and I6 were transformed in E. coli:

• I4 and I6 in E. coli DH5-alpha
• I5 (final part) in E. coli TOP10

1ul of ligation was transformed in 100ul competent cells.

June, 17th

We checked the presence of colonies in plates of I4, I5 and I6 incubated overnight at 37°C. All plates showed colonies. I4 had big, round single colonies. I5 showed big colonies surrounded by small colonies. I6 showed few small colonies and for this reason it was further incubated for 3 hours. Colonies were peaked and inoculated in 1ml LB+antibiotic.

I5-1 and I5-2 were inoculated both in LB+Amp and LB+Cm to have a phenotipic assay: in fact I5 should express the Chloramphenicol resistance. All 9 cultures were incubated at 37°C 220 rpm for 6 hours.

250 ml LB and 250ml LB+Cm for low copy plasmids (91,5 ul of Cm from 1000x stock in 250 ml LB).

After six hours, we checked the growth in liquid of our colonies.

• I4-1 and I4-2 were all grown
• I5-1 and I5-2 were both grown on LB+Amp, but only I5-1 was grown in LB+Cm. I5-2 was negative at this phenotipic assay, so it was discarded.
• I6-1 was apparently not grown, so it was discarded. I6-2 and I6-2 were grown, but the cultures were more limpid than the others, showing that I6 has a slower growth than nomal.

Glycerol stocks were prepared for:

and stored at -80°C.

Remaining cultures were re-filled with 5ml LB+Amp and inoculated at 37°C 220rpm for tomorrow MiniPrep.

June, 18th

All cultures incubated at 37°C 220 rpm overnight were grown. MiniPrep was performed,, with the following quantifications:

June, 21th

June, 22th

June, 23th

June, 24th

June, 25th

Gel run for screening