Part:BBa_K1616005:Experience

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Notebook

Gblocks: ordered of Holin/ Endolysin fragments from IDT

PCR of Holin/Endolysin parts

Aim: Amplification of the part

	MQ Water	RB Buffer	Mg2+	dNTP 10 µM	Primer Fwd 25 µM	Primer Rev 25 µ	DNA	Taq Pol enzyme
Holin - Ensolysin	39 µL	5 µL	1,5 µL	1 µL	1µL pSB1C3 Fwd	1µL pSB1C3 Rev	1 µL	0,5 µL

Digestion of pDawn and Holin/Endolysin

Tube	Buffer 2.1	DNA	EcoRI	Enzyme 2 (0,5 µL)
pDawn	2 µL	12 µL	0,5 µL	NehI
Holin - Ensolysin	2 µL	12 µL	0,5 µL	SpeI

Ligation of Gblocks Holin-Endolysin into pDawn

Tube	Water	T4 ligase Buffer	pDawn	Plasmid	T4 ligase
Holin - Ensolysin	4,6 µL	1 µL	1,4 µL	2,5 µL	0,5 µL

Room temperature, 30min heat kill: 80°C, 20min

Transformation of E.coli DH5 alpha competent cells

Culture liquid: pDawn – H/E 20 mL LB + 20 µL Kan, without light;

Measurement of the OD600 of liquid culture

When the OD600 has reached 0.6 or more, the volume of culture has been divided into 3 erlenmeyers:

• 1 erlenmeyer without light exposition, 37°C
• 1 erlenmeyer with light exposition, 37°C
• 1 erlenmeyer with light exposition, room temperature

Measurement of the OD₆₀₀ every hour
3 culture on 5have shown a bacterial development

Verification if the part

Digestion of pDawn - Holin/Endolysin with EcoRI and PstI

Results

Bacteria expressing pDawn-HE were prepared in a pre-culture until DO600 reached a value between 0,6 and 0,8. Then, one condition was illuminated with white-light and incubated at 37°C, another one was kept in the dark and incubated at 37°C and the last condition was illuminated with white-light and incubated at room temperature. The value of DO was taken regularly to observe the kinetic of growth under each condition.

In absence of light, and at 37°C, bacteria growth was normal but light illumination was enough to slow-down the growth of bacteria. Incubation at room temperature with white-light illumination allowed better folding of HE and had a stronger effect on the growth of bacteria.

User Reviews

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