Team:UNIPV-Pavia/Calendar/August/week5

August, 29th

T9002-ENTERo and ENTERO-RBS were diluted 1:500 in M9 + Amp + Cm12.5 13 ml and 1 ml volume respectively. After six hour growth they were aliquoted in 96-well microplate (190 μl cultures) with 10 μl of supernatants collected on August, 27th and 10 μl of 3OC₆-HSL.
5 flasks were prepared with 29.6 ml H₂O, 200 μl glycerol 80 %, 8 ml M9 salts 5 x and their pH was adjusted to 5.9, 6.3, 6.7, 6.9 and 7.2; they were autoclaved in order to prepare M9 glycerol supplemented minimal medium.
E43-3 DNA was digested with EcorI and PstI restriction nucleases in order to screen the part length by gel-electrophoresis; it showed the correct band.

Plates incubated on August, 27th were all grown, except for J101-31-1C3; for each of them a colony was picked and inoculated in LB + Cm34, 5 ml, while for J101-E7 two colonies were inoculated in LB + Cm34, 5 ml.
E43-2 was again inoculated in order to screen the insert length with gel electrophoresis.

August, 30th

Glycerol stocks were prepared for E2-1C3, E3-1C3, E4-1C3, E5-1C3, E6-1C3, E7-1C3, E9-1C3, E10-1C3, E11-1C3, J101-E5-1C3, J101-E7-1C3-1 and J101-e7-1C3-2.
DNA of each culture was purified:

Plasmid	DNA (ng/μl)
E2-1C3	90.2
E3-1C3	103.7
E4-1C3	72.4
E5-1C3	78.0
E6-1C3	79.1
E7-1C3	77.8
E9-1C3	73.5
E10-1C3	161.8
E11-1C3	68.8
J101-E5-1C3	216.2
J101-E7-1C3-1	74.7
J101-E7-1C3-2	69.2
E43-2	16.6

Each sample was digested with EcoRI and PstI endonucleases (a mix 13 x was prepared for high copy number plasmid parts):

DNA (μl)	H2O (μl)	EcoRI (μl)	PstI (μl)	Buffer H (μl)	Final Volume (μl)
2	19.5	0.5	0.5	2.5	25

while for E43-2 10 μl were digested. In the afternoon the parts were screened by gel electrophoresis:

All parts showed the correct insert length except for E3-1C3. We decided to pick and inoculate another colony from the plate, named E3-1C3-2.
40 μl CaCl₂ 1M, 800 μl Casamino Acids 10 %, 80 μl MgSO₄ 1M and 1.36 ml thiamine were added to previously autoclaved flasks to prepare M9 at different pHs.
Inoculum of BBa_R0040 in J61002, E3-1 in 5 ml LB + Amp and J101-31 in 8 ml LB + Cm12.5.
Inoculum of ENTERO-4C5 in different pH M9 to test 3OC₆-HSL degradation.

August, 31st

E2-1C3, E4-1C3, E5-1C3, E6-1C3, E7-1C3, E9-1C3, E10-1C3, E11-1C3, J101-E5-1C3, J101-E7-1C3-1, E43-2 and E43-3 DNA samples were prepared and sent to BMR Genomics for sequencing.
ENTERO-4C5 cutlures were diulted 1:200 in a final volume of 5 ml M9. After 2 hours 3OC₆-HSL was added to a final concentration of 100 nM and the first supernatant sample was collected. After 1 hour, 4 hours and 22 hours from 3OC₆-HSL supplementation samples were again collected; supernatants were all stored at -20°C in order to measure 3OC₆-HSL concentration with BBa_T9002 biosensor.
BBa_R0040 in J61002, E3-1 and J101-31 were extracted with MiniPrep kit; here are their quantifications:

Plasmid	DNA (ng/μl)
BBa_R0040 in J61002	79.1
E3-1	63.7
J101-31	9.9

These plasmids were digested with EcoRI and PstI endonucleases to transfer them in pSB1C3:

Plasmid	Kind	DNA (μl)	H2O (μl)	Enzyme 1 (μl)	Enzyme 2 (μl)	Buffer H (μl)	Final Volume (μl)
BBa_R0040 in J61002	Insert	12.5	8	1 EcoRI	1 PstI	2.5	25
E3-1	Insert	15.5	5	1 EcoRI	1 PstI	2.5	25
	Insert	20.5	0	1 EcoRI	1 PstI	2.5	25

In gel electrophoresis all inserts showed the correct length:

After gel-extraction digested DNA was quantified:

Part	DNA (ng/μl)
BBa_R0040 in -j61002 (E-P)	7.9
E3-1 (E-P)	2.8
J101-31 (E-P)	1.5

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)
BBa_R0040 in J61002-1C3	pSB1C3 (E-P)	1.5	BBa_R0040 in J61002 (E-P)	6.5	1	1
E3N-1C3	pSB1C3 (E-P)	1	E3-1 (E-P)	7	1	1
J101-31N-1C3	pSB1C3 (E-P)	1	J101-31 (E-P)	7	1	1

Glycerol stock was prepared for E3-1C3-2 and the culture was pelletted.