Team:Groningen/Notebook/18 August 2009

GVP Cluster
Plates

Showed single colony growth on plates with J61002-Metal Promoter-RFP plasmids, and were stored in the fridge for future preculture growth.


 * → The plates with high and low concentration of transformed cells showed colonies in the expected ratio. On all plates 1 in 20 colonies was dark red, indication of ligation of the original high constitutive promoter back into the J61002 vector, or uncut plasmid which was transformed.

Over Night Cultures


 * → All cultures showed growth, and can be used for plasmid isolation.


 * → By growing colonies on medium with either amp. or kan. as a selection tool to find cultures with correct plasmid it was expected that only the tubes with kan. would show growth. Instead the transformed E.coli TOP10 cells seemed resistent to both antibiotics?



Plasmid Purification

Plasmid isolation was performed on the cultures of E.coli TOP10 containing plasmids pSB3K3 with high, medium and low constitutive promoters and GVP 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

Concentration

Restriction Control

Isolated plasmids were cut with fast-digest enzymes EcoRI and PstI to cut out GVP, and create ~6000bp and ~2700bp fragments.




 * → From left to right: (2x) 1kB ladder, (2x) pSB3K3-J23100-GVP (amp.), (2x) pSB3K3-J23100-GVP (kan.), (2x) pSB3K3-J23109-GVP (amp.), (2x) pSB3K3-J23109-GVP (kan.)


 * → The restriction pattern was not expected (~6000bp and ~2700bp), and can be explained by ligation of vector pSB3K3 to J61035, which gives two EcoRI and one PstI site with ~2700bp, ~2000bp and ~1400bp fragments. The gel purification of GVP cluster must have gone wrong during the cutting of wanted fragment, and the vector instead of GVP has been isolated.


 * → During growth of cells the negative control plate with ampicillin also contained colonies, and can be explained by both amp. and kan. resistence on the double vector ligation product.


 * → New o.n. cultures for isolation of pSB3K3 plasmid with J23100, J23106, and J23109 promoters were made in 6mL LB-amp100 medium.

New Plates

From the plates with E.coli TOP10 containing the plasmid J61002 with three metal promoters infront of RFP, seven colonies of each were plated on a new LB-amp100 plate in a straight line to see if it are red or yellow colonies.

New Over Night Cultures

From each plate with E.coli TOP10 containing the plasmid J61002 with three metal promoters infront of RFP, four colonies were used in 5mL LB-amp100 medium for o.n. culture and isolation of plasmid tomorrow.

Transporters
HmtA curios about the dirty experiment. a 1% gel control will show if expected product is there. Unfortunately there was no product, the primer dimer changed and pcr 1 and 2 remain available. PCR again. Different program. Plus starting an other PCR1 and PCR2 to get new megaprimers.

Metal Accumulation
✅ 1% Agarose (1xTBE) gel with restriction analysis of MBP-ArsR fusion protein plasmids, with selfcloser (1), sample (white colony) (2), sample (red colony??) (3). Expected sizes of positive construct are shown. Samples were digested using PstI & XbaI, ~45 min 37 &deg;C. Sample MBP-ArsR (200) was isolated from red organims, indicating the presence of RFP or similar, this was however not engineered in this plasmid. Therefore a deviant restriction pattern was to be expected. It appears as if two different plasmids were present in this sample. Sample MBP-ArsR (50) was transformed (1 uL) into TOP10 cells and plated out, 50 and 200 uL on LB-agar Amp100.


 * Continue with checking E. coli TOP10 transformants + pSB1AC3-mymT
 * Isolate plasmids of o/n culture (also pMAL-MT-GlpF).
 * Check by restriction analysis:
 * Cut 500ng vector with either by:
 * EcoRI and SpeI --> expected size of fragments is: 213 + 3300
 * or only by PstI --> expected size of fragments is: 150 + 3300


 * Run both the colony PCR and the restriction product on a 1% Agarose gel.
 * From left to right:(upper) 1kb marker, colony PCR on colonies 1-4 (MymT primers), colony PCR on colonies 1-4 (VR/VF2 primers); (lower) digest on 1-4 (EcoRI-SpeI), digest on 1-4 (PstI).
 * As there was no positive (with MymT insert) colony, the colony PCR was redone on 22 more colonies. This also didn't give any positive colony.


 * PCR to amplify fMT and MymT-RBS-pre
 * Use the plasmid pMAL-MT-GlpF (isolated 18/08/09) and PCR product MymT-RBS-pre/suff (14/08/09) to amplify the genes.
 * PCR was done using Phusion DNA polymerase, and the following primers:
 * fMT-RBS FW & fMT-suff RV
 * MymT RV & F1-RBS-ATG FW


 * PCR program with TouchDown 10x (Tm62-50&deg;C and Elongation time 30sec) and Cycles 30x (Tm 58&deg;C).


 * PCR SmtA with new primers
 * Use Phusion polymeras, primers: SmtA-RBS Fw & SmtA Rev, and isolated pET29a-SmtA.
 * The program HmtA-Phusion: 10x TouchDown (Tm 60-50, elong time 1:10), 30 x Cycles (Tm 55)
 * Store in the fridge.

Vectors
✅ No promotors were found on gel (1% and 2%, see below).

Restriction analysis of plasmids engineered to contain metal promotors. Constructs were digested using SpeI & EcoRI, expecting to cut out the ~100 bp that is the promotor. Gel is 2% agarose (1xTBE). DNA marker is 1 Kb.

Dry
Jasper worked on cleaning up the Mathematica notebooks for Meng2004 and Kostal2004, the newly derived rate constants for Meng2004 were put on the Wiki (on the transport page).