Team:UNIPV-Pavia/Calendar/August/week5

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UNIPV TEAM 2011

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AUGUST: WEEK 5

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.

Small size gel

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:

Medium size gel

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:

Small size gel

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.