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Team:SYSU-China/file/Project/Notebook/Labnote/M13.html
From 2014.igem.org
Contents |
M13·LABNOTE
May
PLAN:
M13 phage ssDNA isolation.
Study Ph.D.Phage Display Library in details.
SUM UP:
ER2738 enlarging culture and M13 phage ssDNA isolation.
ER2738 host amplification to extract M13KE RF DNA from bacteria cells using plasmid extraction kit.
June
PLAN:
M13 infection.
M13KE extraction .
white-blue plaque selection.
SUM UP:
Use M13 phage to infect ER2738.
Use plasmid extraction kit to extract M13KE infected cells.
July
PLAN:
Amplify the host ER2738.
Try the effect of infection time.
Restriction Analysis to verify the possible M13KE plasmids and pure M13KE plasmids.
Use the remaining samples as PCR templates to get gene II and gene VIII, maybe gene III.
SUM UP:
The extraction product is kind of DNA mixture, which probably containing certain amount of M13KE plasmids.
The sequencing result using M13R.
Get gene II, gene III and gene VIII, construct gene II and VIII into pSB1C3 vector.
August
PLAN:
Confirm pSB1C3-gV + FLAG & pSB1C3-gVIII+FLAG by sequencing.
M13dZ PCR.
SUM UP:
Get pSB1C3-gV+FLAG & pSB1C3-gVIII+FLAG plasmid, though with concentration around 100ng/uL, 50uL in total.
Got M13dZdpIII.
September
PLAN:
pSB1A1-CmR cassette biobrick.
Western Blot
SUM UP:
Get pSB1A1-CmR cassette biobrick.
Data shows that there is no obvious g2p protein expression in ER2738.
Anti-FLAG signal of Western-blot is in wrong protein mass.
October
PLAN:
Perform rescue experiment.
Test the induction efficiency of the pBAD promoter in ER2738.
SUM UP:
Rescue experiment is performed. ER2738 is transformed pSB1C3-araC-pBAD-RBS-pIII-FLAG and M13KdZdpIII at the same time.
Exogenous pIII expression can be induced by Ara.
M13 DNA can be detected in all filtered supernatants.
While detecting the gII, there is no significant difference between the induced or not induced groups of the rescue experiment.
pVIII DNA failed to be detected in rescue experiment groups.
There is virus DNA detected in all groups' supernatant.
The expression of pIII-FLAG protein in co-transformed phage and rescue plasmids has been confirmed.
