Team:UNIPV-Pavia/Calendar/September/settimana5

SEPTEMBER: WEEK 5

I62 and MyYeast screening.

I62-e was positive, so we made glycerol stock and stored it at -80°C.

Protein electrophoresis for phasins (Fede)

September, 28th

Tecan Test Giacomo

Miniprep and quantification for:

• I1: 59,6 ng/ul
• I4: 109,9 ng/ul
• I53: 160,3 ng/ul
• <partinfo>BBa_J04450</partinfo>-1:55,9 ng/ul
• <partinfo>BBa_J04450</partinfo>-2: 47 ng/ul

ON digestion of:

• I1 (E-S)
• I4 (E-S)
• I53 (E-X)
• <partinfo>BBa_J04450</partinfo>-1 (E-P)
• <partinfo>BBa_J04450</partinfo>-2 (E-P)
• MyYeast-3 (DNA already available) (E-S)
• <partinfo>pSB4C5</partinfo> (DNA already available) (E-P)

September, 29th

Colony PCR for 6 newly picked colonies.

I55 colony PCR screening.

No positives nor this time.

Gel run for all digested parts

Digestions.

and cut/gel purification for:

• 4C5 (E-P): 19,3 ng/ul
• My Yeast 3 (E-S): 20,8 ng/ul
• I1: 3,8 ng/ul
• I4 (E-S): 14,2 ng/ul
• I53 (E-X): 21,6 ng/ul
• <partinfo>BBa_J04450</partinfo> (pLac-RBS-mRFP-TT) (E-P): 5,6 ng/ul
• <partinfo>pSB1C3</partinfo>: 10 ng/ul

Ligation of:

September, 30th

Tecan Test Nico

Transformation of I73 (1ul) into E. coli TOP10. It was plated on LB+Amp 100 ug/ml agar plate.

Trasformation of:

• I70 (myYeast with I4, Ptef1-mOrange-tAdh1) in TOP10 and plated on LB+Amp
• I71 (myYeast with I1, mOrange-tAdh1) in TOP10 and plated on LB+Amp
• I72 (pLac-RFP in pSB4C5) in TOP10 and plated on LB+Cm 12,5

All plates were incubated at 37°C ON

Since I55 was negative again we decided to ligate it again (probably we had a bad digest for vector - I20 - and a good one for insert - we used I38 in other ligations without problems). We diegested already miniprepped DNA for three hours with 1 ul of enzymes:

• I20: (E-X)

Gel run/cut/extraction/quantification

I20 E-X digest.

I20 E-X: 13,4 ng/ul

We repeated I55 ligation using newly digested vector and already digested I38 (E-S)

I55: I38 (E-S) + I20 (E-X)

Inoculum into 5ml LB+Amp of:

• I31
• I35
• I37
• I57
• I60
• INTEIN
• 3x <partinfo>BBa_J04450</partinfo> (to take <partinfo>pSB1C3</partinfo>, so <partinfo>pSB1C3</partinfo> from now on)

Cultures were let grow ON at 37°C, 220 rpm.

Inoculum of E. coli TOP10 in 1,5 ml LB, grown ON at 37°C 220 rpm. Tomorrow we will prepare competent cells!!

Inoculum of:

• S1 in 500ul M9+Cm 12,5
• I47-S1 in 500ul M9+Amp+Cm 12,5
• GFP-S1 in 500ul M9+Amp+Cm 12,5
• RBS32 in 500ul M9+Amp

Cultures were grown ON at 37°C 220 rpm.

Tomorrow we will test the ability of these strains to produce GFP and to lyse when HSL is added.

October, 1st

This morning, we checked tranformed plates:

• I72 showed NO colony!! :/ we will repeat this ligation next week
• I70 and I71 showed colonies, 5 for each plate were picked and inoculated in 1ml LB+Amp to prepare glycerol stocks and for screening.
• I73 showed colonies; six of them were picked and inoculated into 5 ml LB+Amp and let grow ON at 37°C, 220rpm in order to do E-P screeening the following day.

Transformation of I55 into E. coli DH5-alpha. It was plated on LB+Amp 100 agar plate and let grow ON at +37°C.

Miniprep and Nanodrop quantification for:

• I31: 76,1 ng/ul
• I35: 66,8 ng/ul
• I60: 279,7 ng/ul

(they were also sent sequencing)

• INTEIN: 123,8 ng/ul
• <partinfo>pSB1C3</partinfo>-1: 104,8 ng/ul
• <partinfo>pSB1C3</partinfo>-2: 141,2 ng/ul
• <partinfo>pSB1C3</partinfo>-3: 113,5 ng/ul
• I37: 174,9 ng/ul

Digest:

• <partinfo>pSB1C3</partinfo>: E-P
• <partinfo>pSB1C3</partinfo>: E-S
• <partinfo>pSB1C3</partinfo>: X-P
• INTEIN: E-P
• I60: E-S
• I31: S-P
• I35: E-P
• I35: E-X
• I37: E-X
• I57: E-X

Gel run/cut/extraction/quantification:

Intein, I31, I35, I37, I57, I60 digestions

<partinfo>pSB1C3</partinfo> digested E-P (both vector and insert were kept), E-S, X-P.

• I37 (E-X): 20,4 ng/ul
• I57 (E-X): 17,6 ng/ul
• I60 (E-S): 9,5 ng/ul

They were stored at -20°C.

• I31 (E-S): 5,4 ng/ul
• I35 (E-P): 1,0 ng/ul ;-(
• I35 (E-X): 14,4 ng/ul
• <partinfo>pSB1C3</partinfo> (E-P): 14,3 ng/ul
• <partinfo>pSB1C3</partinfo> (E-S): 15,2 ng/ul
• <partinfo>pSB1C3</partinfo> (X-P): 13,9 ng/ul

ON ligation of:

• I74: I31 (E-S) + I35 (E-X)
• INTEIN_C3: INTEIN (E-P) + <partinfo>pSB1C3</partinfo> (E-P)

October, 2nd

PCR from colony for I55 (eleven colonies were picked).

Gel run:

I55 colony PCR screening.

We decided to make glycerol stock for I55-1. Inoculum into 5 ml LB+Amp of the positive culture that was let grow ON at 37°C, 220 rpm.

Miniprep and quantification for I73-1/2/3/4/5/6:

• I73-1: 154 ng/ul
• I73-2: 177 ng/ul
• I73-3: 154,7 ng/ul
• I73-4: 145,5ng/ul
• I73-5: 152,6 ng/ul
• I73-6: 154,2 ng/ul

1,5 hours E-P digest (for screening) and gel run

I70, I71, I73, A9_4C5, A99_4C5 screening.

As you can see they are (except for I73-6) all positive. So we stocked I73-1 and stored it at -80°C.

Transformation of I74 and INTEIN_C3 (1ul) in E. coli TOP10. They were plated on LB+Amp100 and LB+Cm34 agar plates respectively. They were let grow ON at +37°C.

Inoculum of I55-1 into 5 ml LB+Amp. ON growth at +37°C, 220 rpm.

October, 3rd

Glycerol stock for:

• I55-1
• IC-1/2/3/4-4C5

Pick of a single non-red colony of INTEIN_C3 and inoculum into 5 ml LB+Cm34. Sample was let grow ON at +37°C, 220 rpm.

Miniprep and quantification of:

• I55-1: 57,4 ng/ul
• I55-1bis: 79,6 ng/ul

I55-1bis was digested E-S for about three hours and than gel run/cut.

Colony PCR for I74-1..6.

Gel run for PCR samples and I55 (E-S) and cut for this one.

I74 screening and I55 E-S cut.

Very bad PCR. We performed a massive PCR from colony during the night (I74-7..24), we ran it the following day.

Gel extraction and quantification:

• I55 (E-S): 5,0 ng/ul

Ligation of I55 (E-S) with already available DNA:

• I75: I55 (E-S) + I37 (E-X)
• I77: I55 (E-S) + I57 (E-X)

Inoculum of I35 (last quantification after gel extraction was too poor; we will digest it again E-P) and I54 into 5 ml LB+Amp: ON growth at +37°C, 220 rpm.