Difference between revisions of "Part:BBa K1616005:Experience"

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Revision as of 00:48, 23 September 2015

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Please enter how you used this part and how it worked out.

Applications of BBa_K1616005

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

Results

Graph-HE.png
Graph-150915 2.png
Graph-150915 3.png


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

UNIQ42225ee873bfaf09-partinfo-00000000-QINU UNIQ42225ee873bfaf09-partinfo-00000001-QINU