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Team:Wageningen UR/Journal/week26
From 2012.igem.org
Week 26: 22 october - 28 october
GFP modification
22 October
- sent bricks BBa_K883704 and BBa_K883705 in for sequencing (single read using the reverse sequencing primer)
-> the bricks have the expected sequence
- seperated colonies containing BBa_K883700 and BBa_K883701 (GFPcoil and GFPcoil with a His tag in pSB1C3) from the plate of 21.october a second time
23 October
- colony PCR on the seperated colonies containing the faulty biobricks BBa_K883700 and BBa_K883701 to check wheather there are colonies present with the correct insert
-> there was no insert present in the picked colonies
- grow batches BBa_K883702 in BL21, BBa_K883703 in JM109, BBa_K883704 in DH5α, BBa_K883705 in DH5α
24 October
- plate BBa_K883702 in BL21, BBa_K883703 in JM109, BBa_K883704 in DH5α, BBa_K883705 in DH5α on plates containing IPTG together with a control that does not produce fluorescence
- checked fluorescence of JM109 cultures (from a plate) containing BBa_K883702 and BBa_K883703 with fluorescence microscopy
-> fluorescence could be seen in both cultures; a negative control (JM109 without a GFP encoding plasmid) showed no fluorescence; the positive control (JM109 containing Bba_I13522 - GFP with a constitutive promoter) showed strong fluorescence
Figure 1: fluorescence microscopy pictures of a negative control (JM109 without a GFP encoding plasmid); JM109 cultures containing the bricks BBa_K883702 GFP-coil and BBa_K883703 GFP-coil with His tag and a positive control (JM109 containing Bba_I13522 - GFP with a constitutive promoter) '
PLRV
We continued our VLP formation attempt with the purification protocol. On thursday, we went to the Wageningen Electron Microscropy Centre across town to see if we had any PLRV VLPs. We couldn't find any.
The sample from the adjusted protocol (skipped a washing step for fear of losing product) was too polluted to see anything. The normal sample contained many vesicles, but no VLPs.
