Part:BBa_K1616005:Experience

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

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

UNIQ5656548a66f06aa0-partinfo-00000000-QINU UNIQ5656548a66f06aa0-partinfo-00000001-QINU