Team:UNIPV-Pavia/Calendar/July/settimana4

JULY: WEEK 4

July, 19th

TECAN test showed that no RFP was produced from our parts, so all parts are potentially correct! For this reason we decided to sequence:

• I14-1 (Forward)
• I16-1 (Forward)
• I17-1 (Forward)
• I18-1 (Forward)
• I19-1 (Forward)

We also sequenced:

• I74C5-2 (Forward and Reverse)
• I84C5-2 (Forward and Reverse)
• I12-2 (Forward and Reverse)

These samples were prepared for sequencing (DNA was essicated) and sent to BMR genomics.

Trasformation of RING into:

• BW25141 (pir+)
• BW25142 (pir116)
• BW23474 (pir116)
• DH5alpha
• MG1655

Cultures were plated on:

• BW2514: Cm 34ug/ml
• BW25142: Cm 34ug/ml
• BW234741: Cm 34ug/ml
• DH5alpha: Cm 12,5ug/ml
• MG1655: Cm 12,5ug/ml

Inoculum of:

• I3-1
• I10-1
• I12-2
• I14-1
• I17-1
• <partinfo>BBa_J23110</partinfo>

in 5ml LB+Amp. Cultures were grown ON 37°C 220 rpm.

BW23473 arrived from Yale University on a paper disk. It was grown ON in 5ml L, at 37°C, 220 rpm.

July, 20th

BW23474 transformed with <partinfo>BBa_J72007</partinfo>

DH5alpha transformed with <partinfo>BBa_J72007</partinfo>

Results for plates incubated ON, 37° C:

• BW23474 (pir116): showed colonies
• BW25141 (pir+): showed colonies
• BW25142 (pir116): showed colonies
• DH5alpha: didn't show colonies
• MG1655: didn't show colonies (even if there were a very few colonies that we suppose integrated the resistance of RING to survive - or the plate antibiotic wasn't homogeneous)

BW23474 transformed with RING	BW25141 transformed with RING	BW25142 transformed with RING
DH5alpha transformed with RING	MG1655 transformed with RING

Single colonies were picked from plates and grown in LB+Cm at proper concentration. MG1655 colonies were let grow in LB+Cm to check if they integrated the Cm resistance of RING.

MiniPrep was performed on cultures incubated yesterday, with following yields:

We retrieved from our freezer the following MiniPreps, with quantifications:

Digestion of:

These parts were gel run/cut:

4C5, I12-2 and I10-1 couldn't be extracted from gel, because bands were insignificant. For this reason we decided not to perform ligations involving I12-2 and I10-1 today. Luckily we retrieved from our freezer 4C5(E-P), so we could perform following ligations:

• I15new=<partinfo>BBa_J23110</partinfo> (S-P) + I3-1 (X-P)
• I14_4C5=I14(E-P)+4C5(E-P)
• I16_4C5=I16(E-P)+4C5(E-P)
• I17_4C5=I17(E-P)+4C5(E-P)
• I18_4C5=I18(E-P)+4C5(E-P)
• I19_4C5=I19(E-P)+4C5(E-P)

Ligations were incubated overnight at 16°C.

Glycerol stock for BW23473.

Our glycerol was contaminated, so we decided to prepare it again!

BW23474-RING was re-inoculated because it is still not grown.

We incoulated from glycerol stock (3ul in 2ml LB+Amp):

• <partinfo>BBa_J23110</partinfo>
• <partinfo>BBa_J23118</partinfo>
• <partinfo>BBa_J23116</partinfo>
• <partinfo>BBa_J23114</partinfo>
• <partinfo>BBa_J23106</partinfo>
• <partinfo>BBa_J23105</partinfo>
• <partinfo>BBa_J23101</partinfo>
• <partinfo>BBa_J23100</partinfo>
• <partinfo>BBa_B0033</partinfo>

in order to perform a TECAN test tomorrow.

Inoculum of MC1061 in LB, PBHR68 in LB+Amp and <partinfo>BBa_T9002</partinfo> in LB+Amp to prepare competent cells tomorrow.

July, 21st

Tecan Test

This morning all cultures were grown:

• BW25141-RING
• BW25142-RING
• BW23474-RING
• BW23474-RING re.inoculated yesterday night (thrown away because unuseful)
• MG1655-1
• MG1655-2
• MG1655-3
• MG1655-4

For these 7 seven cultures glycerol stocks were prepared and stored at -80°C. Remaning 5 ml were used to perform MiniPrep.

After MiniPrep, purified DNA was quantified with NanoDrop.

Digestion of:

Today we also prepared competent cells for cultures inoculated yesterday:

• <partinfo>BBa_T9002</partinfo> (already resistent to Ampicillin, grown in LB+Amp)
• MC1061 (grown in LB with no antibiotic)
• PBHR68 (already resistent to Ampicillin, grown in LB+Amp)

In the afternoon, we transformed our ligations: Trasformation of ligations in:

July, 22nd

July, 23rd

July, 24th

Efficiency of transformation:

July, 25th