Part:BBa_K1616005:Experience
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Applications of BBa_K1616005
Notebook
Gblocks: ordered of Holin/ Endolysin fragments from IDT
BBa_K1616005 has been characterized into pDawn plasmid, however, the submitted part was into pSB1C3
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 |
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Holin - Ensolysin |
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Digestion of pDawn and Holin/Endolysin
Tube |
Buffer 2.1 |
DNA |
EcoRI |
Enzyme 2 (0,5 µL) |
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pDawn |
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Holin - Ensolysin |
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Ligation of Gblocks Holin-Endolysin into pDawn
Tube |
Water |
T4 ligase Buffer |
pDawn |
Plasmid |
T4 ligase |
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Holin - Ensolysin |
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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
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
UNIQ348e3bbb6da3ebe2-partinfo-00000000-QINU UNIQ348e3bbb6da3ebe2-partinfo-00000001-QINU