Team:Groningen/Notebook/28 August 2009

From 2009.igem.org

Wet

GVP Cluster

→ DONE Isolate plasmids from o.n. cultures

→ DONE Restriction analysis of plasmids for correct ligation of GVP behind LAC/pBAD promoter

→ TODO Test control of bouyancy in Saline solution

→ TODO Order synthetic DNA for GVP

→ TODO Test promoter strenght compared to BBa_J23101 promoter (Sven)

→ TODO Enter sequences of constructs to Sandbox

Overnight Precultures

All tubes showed growth of E.coli cells, and must have ampicillin resistance on a plasmid(The tubes differed in cell density though). To be sure of correct ligation products in the plasmids an isolation will be performed, followed by restriction analysis. If positive, the plasmids will be sent for sequencing.

Overnight Plates

The plates grown overnight for glycerol stocks showed single colony growth on the second and third wipe stripes. The plates were stored in the fridge for preculture inoculation on monday (31/8).

→ The colonies for pArsR+RBS showed red colonies, and the pCueO+RBS/pZntR+RBS showed normal yellow/white colonies.

Restriction Analysis

The vector [http://partsregistry.org/wiki/index.php?title=Part:pSB1AC3 pSB1AC3] containing the LAC/pBAD inducible promoters and GVP were cut with PstI and EcoRI to check correct ligation product.

• 5μL plasmid in MQ
• 11μL MQ (end volume of 20μL)
• 2μL Fast digest buffer
• 1μL PstI fast digest enzyme
• 1μL EcoRI fast digest enzyme

Restriction was kept at 37C for 30 min., followed by addition of 6x loading dye and put on ice until used for gel analysis.

→ From left to right: 1kB ladder, pSB1AC3-pLacI-GVP (new, 3x), pSB1AC3-pBad/araC-GVP (new, 3x), pSB1AC3-pLacI-GVP (old, 2x), pSB1AC3-pBad/araC-GVP (old, 2x)

→ Bands are not as expected! The pSB1AC3-pLacI-GVP plasmid should give 6313bp and 3014bp fragments, and pSB1AC3-pBad-araC-GVP should give 7323bp and 3014bp fragments. See the plasmid and part maps below:

• [http://partsregistry.org/Part:BBa_R0010 pLacI]

• [http://partsregistry.org/partsdb/get_part.cgi?part=BBa_K113009 pBad/araC]

• [http://partsregistry.org/Part:pSB1A2 pSB1A2]

• [http://partsregistry.org/Part:pSB1AC3 pSB1AC3]

• [http://partsregistry.org/Part:BBa_I750016 GVP]

→ The lanes with bands at ~2000bp could have been caused by the promoter being in its original plasmid [http://partsregistry.org/Part:pSB1A2 pSB1A2] and GVP ligated behind it. The three lanes for pBad/araC (new) show fragments of ~7000bp corresponding to promoter plus GVP, and have the ~2000bp fragment for pSB1A2.

→ Frans must have a second go at finding the image of his restriction control, which confirms the pBad/araC and pLacI in plasmid [http://partsregistry.org/Part:pSB1AC3 pSB1AC3]. His samples were sent for sequencing and can give conformation in the case of pLacI, because the promoter is only ~200bp there will be room behind it to see if it is the pSB1A2 plasmid!

Observations

→ Tests for bouancy were thought to have been carried out in Saline solution, but instead it was a 1000 times diluted solution. The recipe for Saline is as follows:

• 9 grams NaCl in 1 liter H₂O (1x solution, 0.9% NaCl w/v)
• 90 grams NaCl in 1 liter H₂O (10x soltion)

→ The pBad/araC and pLacI were thought to be transfered to the pSB1AC3 plasmids, and out of the pSB1A2 plasmid. Restriction analysis (used for assembly) showed fragments of the size of pSB1A2, and not pSB1AC3 plasmid. The sequencing results should give desisive prove if it is a correct transfer or re-(self)ligation back into its original plasmid pSB1A2.

New experiment

A 1.5M long glas tube will hold a 550mL colom of 400ml 0,7mM fc iptg induced culture with pNL29 plasmid plus 150 mL saline. We hope to see floating culture that means a transparent bottom layer and a suspension above and some dead cells on the bottom.

Transporters

HmtA

The overnight restriction and the pcr show that only sample 7 can be a correct construct. NcoI will cut 2 times resulting in 2 fragments. One of 1814bp and one of 3261bp. The pcr should result in a grafmetn of 2336bp. this gel does show a convincing cut. but not the expected pcr product.

An other controle PCR F1-REV should give 2222bp

And since it is non-conclusive we start PCR1 and PCR2 again.

Metal Accumulation

Vectors

Dry

Jasper looked at further ways to simplify/analyze our model, no luck so far (Laplace transforms and such don't work for non-linear differential equations). It might be possible to show that the equations resulting from the relative abundances have only one solution though (or show that haven't) by looking at the solution as a continued fraction. He also had a look at cleaning up our photographs of the gas vesicles. It should be possible to extract profiles, but when taking new photos reflections should be avoided and if possible the lighting should as uniform as possible.