Team:Groningen/Notebook/25 August 2009

GVP Cluster

 * → Restriction of GVP and pSB1AC3-Lac/pBAD for assembly
 * → Gel purification of wanted fragments and nanodrop
 * → Clone GVP cluster behind Lac and Arabinose promoter for expression tests in pSB1AC3 plasmid
 * → ✅ Send plasmids with MEtal promoters in BBa_J61002 vector for sequencing
 * → ✅ Check MEtal + RBS promoters on gel with restriction (also XbaI/PstI in Zinc promoter)


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

Metal Accumulation
 PCR check of MPB-ArsR fusion protein, two combinations of primers were used; Forward MBP with Reverse ArsR & Forward ArsR with Reverse MBP, giving the indication where proteins are in comparison of each other. Only the first gave the expected product of 1500 bp, indicating that the fusion was as expected MBP-ArsR and nog ArsR-MBP. Sample was send for sequencing.
 * PCR to check MBP-Ars


 * Continue cloning SmtA and fMT in pSB1AC3 + promoters
 * Miniprep pSB1AC3-fMT and SmtA using the Sigma Kit.
 * Send the constructs for sequencing.
 * Digest pSB1AC3-fMT and SmtA with XbaI and PstI, digest pSB1AC3-pLow, pSB1AC3-pLac and pSB1AC3-pBAD with PstI and SpeI.
 * Run the restriction products on a 1% Agarose gel
 * -From left to right: Marker, SmtA 2/X-P, SmtA 3/X-P, fMT 4/X-P, fMT 6/X-P, fMT 6/X-P(only a few uL), pSB1AC3-pLow/S-P, pSB1AC3-pLac/S-P, pSB1AC3-pBAD/S-P, marker.
 * -Expected size: SmtA 900bp, fMT 200bp, pSB1AC3 3300bp, pSB1AC3-pBAD 3500bp.


 * The bands of SmtA 2/X-P were different from SmtA 3/X-P, it seems like an extra restriction site has been inserted into the gene as the fragment sizes are ~600 + ~300.
 * The correct bands were excised and the DNA purified using the Roche Kit.
 * Ligation was done overnight @ 4&deg;C of pSB1AC3-pLow + fMT and SmtA and pSB1AC3-pLac + fMT and SmtA as the concentration of pSB1AC3-pBAD was way to low...

Metal promotors
Cloning of the metal promoters into pSB1AC3 and pSB3K3 has stopped

Dry
Jasper finally finished simplifying the differential equations and they are now presented in neat tables on our modelling page. It turned out that it is easiest to work with differential equations for the totals of substances in combination with relative abundances of the different forms of the substances.