Team:UNIPV-Pavia/Calendar/July/settimana3

July, 11th

Digestions of previously purified plasmids were performed for ligations:

Plasmid	Kind	DNA (μl)	H2O (μl)	Enzyme 1 (μl)	Enzyme 2 (μl)	Buffer H (μl)	Final Volume (μl)
E1-2	Insert	18	2.5	1 XbaI	1 PstI	2.5	25
E2-2	Insert	18	2.5	1 XbaI	1 PstI	2.5	25
E3-1	Insert	18	2.5	1 XbaI	1 PstI	2.5	25
E4-2	Insert	18	2.5	1 XbaI	1 PstI	2.5	25
E5-2	Insert	18	2.5	1 XbaI	1 PstI	2.5	25
E6-1	Insert	11	9.5	1 XbaI	1 PstI	2.5	25
E7-2	Insert	18	2.5	1 XbaI	1 PstI	2.5	25
E8-3	Vector	20.5	0	1 SpeI	1 PstI	2.5	25
BBa_R0040	Vector	14.5	6	1 SpeI	1 PstI	2.5	25
BBa_C0261	Insert	18	2.5	1 XbaI	1 PstI	2.5	25
BBa_I13507	Insert	10	10.5	1 XbaI	1 PstI	2.5	25
BBa_B0034	Vector	10	10.5	1 SpeI	1 PstI	2.5	25

Reactions were incubated at 37°C for three hours while two medium-size agarose gels were prepared according to protocols.

In the afternoon gel electrophoresis was performed.

As shown, in figure all clones were positive, so we cut and purified the bands of interest.

After gel extraction, digested DNA was quantified:

Part	DNA (ng/μl)
E1 (X-P)	4.2
E2 (X-P)	3.8
E3 (X-P)	4.1
E4 (X-P)	3.7
E5 (X-P)	5.6
E6 (X-P)	7.8
E7 (X-P)	6.4
E8 (S-P)	3.4
BBa_C0261 (X-P)	4.8
BBa_R0040 (S-P)	7.9
BBa_B0034 (S-P)	11.0
BBa_I13507 (X-P)	6.8

Then ligations were performed in a final volume of 10 μl:

Ligation Name	Vector	Vector volume (μl)	Insert	Insert volume (μl)	Buffer (μl)	T4 Ligase (μl)
E9	BBa_B0030 (S-P)	1	E1 (X-P)	7	1	1
E10	BBa_B0031 (S-P)	1	E1 (X-P)	7	1	1
E11	BBa_B0032 (S-P)	1	E1 (X-P)	7	1	1
E12	BBa_B0034 (S-P)	1	E1 (X-P)	7	1	1
E13	BBa_R0040 (S-P)	1.5	E2 (X-P)	6.5	1	1
E14	BBa_R0040 (S-P)	1.5	E3 (X-P)	6.5	1	1
E15	BBa_R0040 (S-P)	1.5	E4 (X-P)	6.5	1	1
E16	BBa_R0040 (S-P)	2	BBa_C0261 (X-P)	6	1	1
E17	E8 (S-P)	4.5	E5 (X-P)	3.5	1	1
E18	E8 (S-P)	5	E6 (X-P)	3	1	1
E19	E8 (S-P)	5	E7 (X-P)	3	1	1
E20	E8 (S-P)	5	BBa_I13507 (X-P)	3	1	1
E21	BBa_R0040 (S-P)	2	E5 (X-P)	6	1	1
E22	BBa_R0040 (S-P)	2.5	E6 (X-P)	5.5	1	1
E23	BBa_R0040 (S-P)	2	E7 (X-P)	6	1	1

Ligations were incubated ON at 16°C.

July, 12th

E9, E10, E11, E12, E14, E15, E17, E18, E19, E20, E21, E22 and E23 were transformed in 100 μl of TOP10 competent cells according to protocols, while E13 and E16 ligations were transformed in 100 μl of MGZ1 competent cells. Plates were incubated ON at 37°C.

July, 13th

All plates showed a lot of colonies, except for E13, E16 (one colony),while for E14, E15 no colonies were observed (probably the parts gave too high metabolic burden that prevents cells growth or because of low transformation efficiency of MGZ1 strain). Because of RFP, E21 was red, E22 and E23 a little bit less. Two colonies (where possible) were picked for each plate.
500 ml of LB with Ampicillin were prepared.
Another ligation was performed:

Ligation Name	Vector	Vector volume (μl)	Insert	Insert volume (μl)	Buffer (μl)	T4 Ligase (μl)
BBa_R0040 in BBa_J61002	BBa_R0040 (S-P)	3.5	BBa_J23101 (X-P)	4.5	1	1

Glycerol stocks were prepared for every culture, except for E11-1, E21-2, E23-2 because they were not sufficiently grown.

July, 14th

Glycerol stocks for E11-1, E21-2 and E23-2 were prepared. Cultures grew overnight; plasmid purification and quantification were carried out:

Plasmid	DNA (ng/μl)
E9-1	114
E10-1	72
E11-1	78.5
E12-1	151.7
E13-1	133
E16-1	134.4
E17-1	27.8
E18-1	25.5

A 25 x mix was prepared in order to perform PCR on E9-1, E9-2, E10-1, E10-2, E11-1, E11-2, E12-1, E12-2, E17-1, E17-2, E18-1, E18-2, E19-1, E19-2, E20-1, E20-2, E21-1, E21-2, E22-1, E22-2, E23-1, E23-2:

H2O (μl)	Buffer 10x (μl)	MgCl2 (μl)	VF2 (BBa_G00100) μl	VR (BBa_G00101) μl	dNTPs (μl)	Taq polymerase (μl)
450	62.5	25	12.5	12.5	12.5	25

24 μl were aliquoted in each tube, together with 1 μl of each liquid culture. PCR cycles parameters were set as follows:

• 94°C 30 seconds (denaturing)
• 60°C 1 minute (annealing)
• 72°C 2 minutes (elongation)

35 cycles were performed.

In order to screen the DNA of the only two colonies of E13 and E16, a digestion for each ligation was performed:

Plasmid	DNA (μl)	H2O (μl)	Enzyme 1 (μl)	Enzyme 2 (μl)	Buffer H (μl)	Final Volume (μl)
E13-1	3	18.5	0.5 EcoRI	0.5 PstI	2.5	25
E16-1	3	18.5	0.5 EcoRI	0.5 PstI	2.5	25

Reactions were incubated at 37°C for three hours.
BBa_B0032 was transformed in 100 μl of MGZ1 competent cells to test if there was any problem with their transformation efficiency, while BBa_R0040 in BBa_J61002 was transformed in 200 μl of TOP10 competent cells. Plates were incubated ON at 37°C.
Two medium size gels were prepared.

In the afternoon gel electrophoresis was performed:

As shown in figure, all clones were positive, except for E9-1, E17-1, E17-2, E18-1, E18-2, E19-1 which didn't show any DNA band. E9-2, E17-2, E18-2, E19-2, E20-2, E21-1, E22-2, E23-1 plasmid purification was carried out, in order to prepare samples for sequencing.

Plasmid	DNA (ng/μl)
E9-2	66
E17-2	28
E18-2	26.3
E19-2	39.1
E20-2	22.1
E21-1	143.1
E22-2	70.4
E23-1	80.9

E9-2, E10-1, E11-1, E12-1, E13-1, E16-1, E19-2, E20-2, E21-1, E22-2 and E23-1 glycerol stocks were preserved, while the others were thrown away. Cells harbouring E9-2, E10-1, E11-1, E12-1, E13-1, E16-1, E21-1, E22-2, E23-1 were inoculated in 5 ml LB + Amp for next week ligations.

July, 15th

The plate containing MGZ1 transformed with BBa_B0032 showed a lot of colonies with satellite cells; a colony was picked for BBa_R0040 in BBa_J61002 as the plate showed red colonies and it was inoculated in 5 ml of LB+Amp and gorwn at room temperature.
Liquid cultures grew until saturation.

In order to screen again E17-1, E17-2, E18-1 and E18-2 ligations, digestions with EcoRI and PstI endonucleases were carried out:

Ligation Name	DNA (μl)	H2O (μl)	Enzyme 1 (μl)	Enzyme 2 (μl)	Buffer H (μl)	Final Volume (μl)
E17-1	6	15.5	0.5 EcoRI	0.5 PstI	2.5	25
E17-2	6	15.5	0.5 EcoRI	0.5 PstI	2.5	25
E18-1	6	15.5	0.5 EcoRI	0.5 PstI	2.5	25
E18-2	6	15.5	0.5 EcoRI	0.5 PstI	2.5	25

Digestions were incubated at 37°C for three hours, while a small size gel was prepared; in the afternoon gel electrophoresis was carried out:

As three out of four clones were correct, E17-2 and E18-2 were kept.
E9-2, E10-1, E11-1, E12-1, E13-1, E16-1, E19-2, E20-2, E21-1, E 22-2 and E23-1 DNA was sent to BMR genomics for sequencing.
Plasmid purification was performed on liquid cultures:

Plasmid	DNA (ng/μl)
E9-2	87.0
E10-1	102.5
E11-1	242.8
E12-1	105.6
E13-1	163.6
E16-1	246.1
E21-1	129.5
E22-2	108.5
E23-1	102.3

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