Team:Groningen/Notebook/31 August 2009

GVP Cluster
Concentrations

Restriction for Assembly

The vector pSB1A2 containing the pLacI/GVP and pBad/araC/GVP composite parts were cut with PstI and EcoRI to create correct ends for insert into pSB1AC3, which was also cut with EcoRI and PstI.

Restriction was kept at 37C for 30 min. and put on ice until used for gel purification.

Purification




 * → From left to right: 1kB ladder, pSB1A2-pLacI-GVP (2x) ,Empty Slot, pSB1A2-pBAD-AraC-GVP (2x), Empty Slot, pSB1AC3-H (2x)

A "Agarose Gel DNA Extraction Kit" Standard Protocol from Roche Applied Science.


 * → In step ten, 30μL MQ was added to the dry pellet and incubated at room temperature.

Concentrations

Ligation

A total amount of vector of 125-200ng was used (promoter-GVP) in a 1:2 and 1:3 ratio with insert.

(1:3)
 * 3 uL Ligase buffer
 * 1 ul T4 Ligase
 * 8 uL plasmid pSB1AC3 digested with EcoRI and PstI
 * 8 uL insert pBad-araC-GVP restricted with EcoRI and PstI

(1:2)
 * 3 uL Ligase buffer
 * 1 ul T4 Ligase
 * 11 uL plasmid pSB1AC3 digested with EcoRI and PstI
 * 5 uL insert pLac-GVP restricted with EcoRI and PstI

Incubate:
 * 25°C 50min.
 * kept on ice for 10min.

Tranformation
 * add 10uL of the ligation product to 50uL competent E.coli TOP10 cells.

Incubate:
 * 30 min @ ice
 * 90 sec 42°C
 * 2 min @ ice
 * add 800uL LB-medium
 * incubate for 1 h at 37°C
 * plate on LB-amp100 plates


 * → Positive control was BBa_J61002-J23101 plasmid, and negative control was MQ.

Observations


 * → The pLacI turned out to be in the pSB1A2 plasmid instead of pSB1AC3 (Frans isolated self ligation products, and ended up with the original biobrick from the registry). And the plasmid I used turned out to be the pLacI-RBS combination, which gives me the pLacI-RBS(new)-RBS(from GVP)-GVP construct!!! New ligation of GVP to pLacI must be done to eliminate the additional RBS site.


 * → The sequence results of the pBad/araC in pSB1AC3 contained N at each 10th position, and therefor it is hard to tell if the pBad/araC sequence is correct. The same problem was spotted here as with the pLacI, there is a additional RBS site present!!!


 * → The biobrick plasmid with pBad/araC seemed to be misplaced by the persons working with it, and no glycerol stock was made. There is a slim change a few of the plates have been stored in the fridge.

Overnight Plates


 * Two plates with pSB1A2-pBad/araC-GVP and pSB1A2-pLacI-GVP for glycerol stocks were stored in the 37C stove.


 * Four plates with pSB1AC3-pBad/araC-GVP and pSB1AC3-pLacI-GVP transformation product were stored in the 37C stove for colony growth. (along with positive and negative control plates)

Overnight Cultures


 * Four tubes with pNL29, GVP, pLacI and pBad/araC (first three from stock, last one was from plate with satelite colonies, amp. should get rid of unwanted cells).


 * Three tubes with pZntR+RBS, pArsR+RBS and pCueO+RBS in J61002 were grown for glycerol stocks.


 * One tube with pNL29 from stock for growth control.

Transporters
HmtA

The cloning strategy will be changed. First we will make a PCR2 (261bp) and a PCR1.2 (1122bp) where F2-mut1rc plus Rev-mut2rc will generate a HmtA where we will add a prefix via a PCR with F1-REV. And than ready for cloning. But first we wait for a kit.

An aditional PCR started to get more megaprimer. This worked (1122bp and 261bp) and therefore there is an other sample ready for gelextraction.

Metal Accumulation
Colony PCR of SmtA with low constitutive or IPTG inducible promotor.

Dry
Jasper looked at extracting lightness profiles from photographs. The results will be in SVN tomorrow (there was a little trouble with uploading the files over the wireless network). So far it mostly looks like it could provide a cosmetic advantage, in the sense that graphs might be easier to read than vague photographs (especially in a presentation).