Cloning of FlhB promoter

Protocol

• Preparation of the template :

Resuspension of 1 colony E.coli K12 strain MG 1655 in 100µl of water.

• Preparation of PCR mix :

1µl of dNTP
10µl Buffer Phusion 5X
2.5µl O 108
2.5µl O 109
1µl Template
1µl Phusion
33µL pure water

Result

[[Image:KR00019b.jpg| thumb| Vérification of pFlhB]

Construction of pFlgA - YFP tripart (+/- LVA)

Aim : Construction of "pFlgA-RBS-YFP-dbl ter" (pFlgA-E0430/E0432)

Digestion

Gel Extraction

Protocol [[Image:KR00019b.jpg| thumb|Gel Extraction of D166-D167]

Measurement of the concentration of D166 & D167 purified

Protocol (it's same that for Miniprep)

Ligation

Protocol

Screening of the cloning of E0240 and FlhDC+promotor

We obtained single colonies with the dilution 100.
13 clones were analysed by PCR

PCR screening

reaction mixture (25 µL)

• 12,5 µL Quick load PCR Mixture 2X
• 0,5 µL O18
• 0,5 µL O19
• 11,5 µL water

PCR screening programm

• elongation time: 1 min 30
• primers: O18 and O19
• positive control: S158 (pSB3K3)
• negative control: no template

Electrophoresis

Gel n°1 (E0240 screening)

Gel n°2 (FlhDC+promotor screening)

• 1% agarose gel
• 10 µL loaded

Results:

• The clone of E0240 (S159.1) always have several bands amplified by PCR. It might contain different plasmids.
• The clone of FlhDC+promotor (S161.1) don't have the correct size band. It also doesn't have the insert in the plasmid.

Construction for synchronization

Ligations

Protocol

Ligation Name	Description	Vector Name	Volume vector (µL)	Insert Name	Volume insert(µL)
L158	rbs-TetR + gfp tripart	D110 (BV)	2	D131 (BI)	2.89
L159	rbs-lasI + Double terminator	D125 (FV)	2.08	D109 (FI)	1.15