Team:Groningen/Notebook/3 September 2009

Wet
LB-medium is badly needed!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

GVP Cluster
 EM pictures 


 * → ✅ Today we will try to get some Electon microscopy pictures of the gas vesicle producing bacteria from the ON cultures.

Orinigal pNL29 iptg-GVP and the biobricks ars-GVP and


 * → Choose colonies from plates for growth of E.coli TOP10 with GVP behind LAC/pBAD (+RBS) promoter in pSB1AC3


 * → Make glycerol stocks of pBad/araC, pBad/araC+RBS+GVP, and pLacI+RBS+GVP (both in pSB1A2 and pSB1AC3 plasmid)


 * → Test control of bouyancy in Saline solution (grow plates with GVP constructs)


 * → ✅ Order synthetic DNA for GVP
 * → Order primer for PstI site removal


 * → ✅ Test promoter strenght compared to BBa_J23101 promoter (Sven)
 * → Enter sequences of constructs to Sandbox

Colonies on Plates


 * → The plates showed normal colony growth.

O.n. precultures


 * → All twelve o.n. precultures showed bacterial growth, and could be used to isolate plasmid. The first four are registry vector pSB2K3 with RFP as reporter (plate 1, 7C) which were transformed on tuesday. The isolated plasmid is used to house all GVP constructs to combine with the vector pSB1AC3 with transporter and accumulation genes.


 * → The next eight tubes contain our own designed biobricks and isolated plasmid will be sent to the registry on friday!



Plasmid Purification

Plasmid isolation was performed on the cultures of E.coli TOP10 containing the above mentioned plasmids with the "Sygma-Aldrich™ GenElute™ Plasmid Miniprep Kit".


 * From each tube 4mL of culture was collected in a 2.0mL cup, and the cells were pelleted by centrifugation for 1 min. at max. speed and the supernatant discarded.
 * Plasmids were eluted with 30μL MQ and stored in the fridge

Concentrations

Restriction for Assembly

The vector BBa_J61035 containing the pArsR, pZntR, and pCueO with GVP composite parts were cut with PstI and EcoRI to create correct ends for insert into pSB2K3, which was also cut with EcoRI and PstI (4x).

Restriction was kept at 37C for 40 min. and put on ice until used for gel purification.



Purification




 * → From left to right: 1kB ladder, pSB2K3 (no.1, no.2, no.3, and no.4), J61035-pArsR-GVP, Empty Slot, J61035-pZntR-GVP, Empty Slot, J61035-pCueO-GVP


 * → The red lines indicate the expected position of the pSB2K3 fragments, ~4400bp for vector and ~1070bp for RFP fragment. The size of the RFP fragment seems to be correct, but the vector part is ~1500bp to small. It can be caused by the slight overloading of the gel, but for the ligation products of tomorrow extra care must be taken to be sure of the vector.


 * → One thing that suggest it must be the vector is its resistance for kanamycin, and the high amount of plasmid after growth with IPTG.

A "Zymoclean(TM) Gel DNA Recovery Kit" standard protocol was used.


 * In step 7 an amount of 10μL MQ was added to elute the DNA fragments.

Concentrations

Metal Accumulation

 * Send MBP-ArsR for sequencing again
 * Send pLow-fMT for sequencing
 * Send pLac-fMT for sequencing

TEM
 Transmission electron microscopy 

The three pictures below show pNL29 induced with iptg for one day. (1) magnification 22.000X (2) magnification 35.000X (3) magnification 260.000X on one vesicle.

In the first two picture we see some light round structures comparable to the structures shown in the Li and Cannon paper. The third picture is what we believe can be a gas vesicles (outside of the cell). Unfortunately these cells have a cellwall. Next week we will make pictures of a protoplast, we hope to reproduce the papers result more clearly.