Ligation, transformation of Vh_quorum

One colony has grown O/N on the Petri dish. This colony was picked in liquid LB media culture with Cm.

Possible explanations for the low amount of colonies:

• pippeting imprecisions
• Strong concentration of Cm
• Competent cells: 106 only

Another ligation was carried out today, using more DNA and another Cm concentration. The ligation was processed as follows: Ligation protocol

A ratio 1:10 of plasmid/insert was chosen:

Competent cells transformation was processed following the transformation protocol.

NB: because of the lack of a right 42°C water bath, the heatshock step was made at 37°C for 40 sec.

2 different plates were made: one at [Cm] = 25 g/L and the other at [Cm] = 12.5 g/L

6 Clones, 2 from CM25 (25-1 and 25-2) and 4 from Cm 12.5 (12-1, 12-2, 12-3 and 12-4 ) that appear on Cm25 and Cm12.5 were grow on plate and on liquid media (5mL) LB + Cm 25 in order to miniprep them with the Miniprep protocol

Digestions were made on the plasmids obtained with the digestion protocol.

In order to confirm the insert, two more digestion were made, with PvuII (which cut once on pSB1C3, and not on the insert) and SacI (which cut once on the insert, and once on pSB1C3) following the digestion protocol

Cloning of Vc_CqsA

The gel extraction from the 08/10 was digested using EcoRI and SpeI with the digestion protocol. The quantities for QSP 100 were:

• 2.5µL of EcoRI
• 2.5µL of SpeI
• 30µL of DNA template
• 10µL of Cutsmart
• 55µL of water mQ

No heat inactivation were made. Instead, a PCR puriffication was processed using Sigma GenEtut^TM PCR Clean Up kit. Afterward, a DNA quantification gel were made containing:

• 2µL of ladder 1kb
• 1µL of loading dye + 5µL of digested DNA
• 1µL of loading dye + 5µL of digested pSB1C3

Consequently, the ligation was carried out and lasted 50min. The followig quantities were used :

• 2µL of ligase buffer
• 1µL of ligase
• 10.8µL of water mQ
• 1.4µL of pSB1C3 digested E/S
• 4.8µL of insert

Finally, Top 10 cells were transformed using the transformation (RbCl-method) protocol. Three aliquots were used:

• 100µL of competent celles + 10µL of DNA: plate A
• 100µL of competent cells + 5 µLof TE buffer + 5µL of DNA: plate B
• 100µL of competent cells + 9µL of TE buffer + 1µL of pSB1C3 non digested (positive control): plate C

After O/N incubation, the followging observation were made on the Petri dishes:

• Plate A: 12 transformants
• Plate B: 19 transformants
• Plate C: cell mat (even with a dilution x10)

Hence, eight transformants from each plate were put on liquid LB-Cm medium for subsequent mini prep. They were named A 1 to 8 and B 1 to 8. The transformants named were minipreped. The resulting DNA solutions were stored at -20°C.

After a quick analysis gel of all the miniprep, without any digestion (data not shown), 6 transformants seems to have the right plasmid lenght: A3, A4, A5, B3, B5, B6. Thus, these transformants were digested with PvuII and E/P to assess the right insertion of Vc CqsA.
Premix for PvuII (for 8 digestions):

• 2 µL of pSB1C3 E/S + 8 µL of PvuII premix
• 2 µL of A3 miniprep + 8 µL of PvuII premix
• 2 µL of A4 miniprep + 8 µL of PvuII premix
• 2 µL of A5 miniprep + 8 µL of PvuII premix
• 2 µL of B3 miniprep + 8 µL of PvuII premix
• 2 µL of B5 miniprep + 8 µL of PvuII premix
• 2 µL of B6 miniprep + 8 µL of PvuII premix

Each tube was put in a water bath at 37°C for 1h. The resulting digestion were put on a 1% agarose gel as follows:

Clones A4 and A5 were named Vc CqsA 1 and Vc CqsA 2 and put in liquid culture to be stored at -80°C the next day.

Solid Bioluminescence assay

Four E. coli precultures were made in LB-Cm (5 mL):

• Vh CqsA Max MG1655
• Vh CqsA Anna MG1655
• Vh CqsA Teo MG1655
• pSB1C3 empty MG1655

Each tubes were complemented with 208 µL of a sterile glucose solution to reach a final concentration of 10 g/L. The tubes were put at 37°C O/N. In addition, two V. harveyi precultures were made:

• LB: Vh WT (5 mL)
• LB: JMH626 (10 mL)

They were put at 30°C O/N. OD of the O/N precultures were taken at 8.06 am. Therefore, fresh LB-Cm-Glc flasks of 10 mL were inoculated to reach OD = 0.1. (final glucose concentration in the medium: 10 g/L). Time of inoculation: 8.32 am. As OD = 0.3 had already been passed at 10.40 am, IPTG induction was made immediatly (final concentration : 0.5 mM). At 1 pm, the cultures were at the end of their exponential phase. Thus, the supernatants could be retrieved following the next steps:

• pipeting of 10 mL of each of the cultures in (x5) eppendorf tube of 2 mL
• centrifugation max speed, 5 min
• pooling of each resulting supernatants
• filtration through a 0.2 µm filter
• storage at -20°C

OD of the V. harveyi O/N precultures were checked again at 11.10 am: JMH626: 4.15, Vh WT:3.11

(x5) LB flasks of 10 mL were inoculated with JMH626 to reach OD = 0.1. In addition, (x1) LB flask of 10 mL was inoculated with Vh WT, also at OD = 0.1. Time of inoculation: 11.28 am. When the OD was around 0.7, each of the 10 mL cultures were centrifugated at 4500 rpm for 6 min. The resulting supernatants were discarded while the pellets were resuspended with 5 mL of fresh LB medium and 5 mL of SN (one flask = one SN). Then, the cultures were put at 30°C. The whole process of resuspension was over at 3.30 pm.

In addition, 80 µL of the resuspended JMH cultures were dropped-off on a Petri dish and put at 30°C O/N. Acting as a landmark of bioluminescence, 80 µL of a Vh WT culture were also put on the Petri dish. For liquid cultures, the positive control showed bright bioluminescence as expected. Meawhile, all the cultures with the clones SN stayed dark. Regular checkings didn't bring more information. The Petri dish incubated O/N at 30°C was observed.

Additional experiments need to be performed to conclud on these bioluminescence essays. Particularly, bioluminescence of JMH626 without any supplementation has to be tested.