Team:UNIPV-Pavia/Calendar/August/settimana1

AUGUST: WEEK 1

August, 2nd

Miniprep and quantification with Nanodrop of:

• I20-1: 98,2 ng/ul
• I20-2: 63,6 ng/ul
• I20-3: 41,5 ng/ul
• I21-1: 45 ng/ul
• I21-2: 45 ng/ul
• I21-3: 54 ng/ul

These samples were prepared and sent (400ng) to BMR Genomics for sequencing.

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The following parts were resuspended from iGEM 2010 Distribution Kit:

• <partinfo>BBa_R0062</partinfo> (Plate 1, Well 6O)
• <partinfo>BBa_K081009</partinfo> (Plate 2, Well 10N)

both in vector <partinfo>pSB1A2</partinfo>.

Transformation (1ul) of the following parts (resuspended/already miniprepped):

Part	Strain	Culture name
pAH123	MC1061	MC123
	MG1655	MG123
<partinfo>BBa_J72008</partinfo>	MC1061	MC008
	MG1655	MG008
<partinfo>BBa_R0062</partinfo>	DH5-alpha
<partinfo>BBa_K081009</partinfo>

Transformed cells were plated on proper LB+Amp agar plates and grown ON at right temperature:

Part	Plate resistance	Temperature
pAH123	Amp 50 ug/ml	30°C
<partinfo>BBa_J72008</partinfo>
<partinfo>BBa_R0062</partinfo>	Amp 100 ug/ml	37°C
<partinfo>BBa_K081009</partinfo>

August, 3rd

Check for plates grown ON: all plates showed colonies.

<partinfo>BBa_R0062</partinfo> plate

<partinfo>BBa_K081009</partinfo> plate

A single colony was picked from <partinfo>BBa_R0062</partinfo> and <partinfo>BBa_K081009</partinfo> plates and inoculated in 1 ml LB+Amp 100 ug/ml and incubated 37°C, 220 rmp for glycerol stock. Cultures left were refilled to 5 ml of proper medium and incubated ON at 37°C, 220 rpm for further screening.

Since plates left showed small colonies they were let grow until late afternoon; than some colonies were picked from each plate and inoculated into a 5 ml LB+Amp 50 ug/ml falcon. Falcon tubes were incubated and shaken ON at 30°C.

MC1061 transformed with pAH123	MC1061 transformed with <partinfo>BBa_J72008</partinfo>
MG1655 transformed with pAH123	MG1655 transformed with <partinfo>BBa_J72008</partinfo>

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PCR from the following colonies (this is a test for the efficiency of our primers synthesized to check attPhi80 E. coli genomic integration and it will be our negative control for future screenings):

• MC1061-1
• MC1061-2
• MG1655-1
• MG1655-2
• Blank (Nothing)

Gel run of amplified DNA showed for every sample the expected distance between primers of a strain with nothing integrated in attPhi80 site (~570 bp), but unfortunately we forgot to take a picture of the gel ;(

August, 4th

Glycerol stocks for MC1061 and MG1655 strains transformed with pAH123 or <partinfo>BBa_J72008</partinfo> helper plasmids.

2 ul of MC1061 bacteria were transferred into 5 ml LB+Amp 50 ug/ml and grown and shaken at 30°C over-day and over-night for re-competentization of the following day.

200 ul of MG1655 cultures were transferred into 100 ml LB+Amp 50 ug/ml and grown and shaken at 30°C for re-competentization of today.

All cultures were miniprepped to check again the presence of helper plasmids.

Samples were digested with SpeI (it cuts twice) for 3 hours and gel run:

• pAH123 digested: 3580 and 2755 bp
• <partinfo>BBa_J72008</partinfo> digested: 2755 and 2437 bp

pAH123 and <partinfo>BBa_J72008</partinfo> screening transformed into MG1655 and MC1061

Samples are positive (right lengths) but unfortunately we got a bad gel run (smearings) so this time we decided to pick two single colonies from each of the plates made on August, 3rd and to inoculate them into 5 ml LB+Amp 50 ug/ml. A total of eight falcon tubes was incubated ON at 30°C, 220 rpm.

We planned to screen them (to check the presence of helper plasmids again) and to re-competentize only the positive ones.

Autoinducibles

August, 5th

Glycerol stock and miniprep of MG1655 and MC1061 cultures incubated for 19 hours at 30°C, 220 rpm. Miniprep was quantified as follows:

• MG123-1: 27 ng/ul
• MG123-2: 40,2 ng/ul
• MG008-1: 20,5 ng/ul
• MG008-2: 26,5 ng/ul
• MC123-1: 21,4 ng/ul
• MC123-2: 17,4 ng/ul
• MC008-1: 33,3 ng/ul
• MC008-2: 38,1 ng/ul

DIgestion for 1 hour with SpeI.

Gel run on medium agarose gel.

pAH123 and <partinfo>BBa_J72008</partinfo> screening (miniprepped from MG1655 and MC1061)

This time gel run succeeded, we chose samples

• MG123-1
• MG008-1
• MC123-1
• MC008-1

to be re-competentizied. So they were inoculated into 5 ml LB+Amp 50 ug/ml and grown and shaken ON at 30°C.

Sequencing for I20 and I21 arrived from BMR, but all samples were wrong; sites X and S of the vector paired and nothing could ligate. So we started a new ligation cycle dephosphorylating the previously gel-extracted vector <partinfo>pSB1A3</partinfo>. New ligations:

• I20-new: Pha-10S-1 (X-S) + <partinfo>pSB1A3</partinfo> (X-S, dephosphorylated)
• I21-new: Pha-SS-1 (X-S) + <partinfo>pSB1A3</partinfo> (X-S, dephosphorylated)

and their negative control:

• C-: <partinfo>pSB1A3</partinfo> (X-S, dephosphorylated)

August, 6th

Resuspension of linker <partinfo>BBa_K105012</partinfo> from iGEM 2010 Distribution Kit.

Transformation of ligations and resuspended DNA

• I20-new
• I21-new
• C-
• <partinfo>BBa_K105012</partinfo>

into 100ul E. coli DH5-alpha.

They were plated on LB+Amp 100ug/ml agar plates

Competentization of colonies selected the previous day:

• MG123 (without '-1' from now on)
• MG008
• MC123
• MC008

August, 7th

Plates of transformed cells were stored at +4°C.