Team:Groningen/Notebook/1 September 2009

From 2009.igem.org

Wet

GVP Cluster

→ DONE Isolate plasmids from o.n. cultures

→ TODO Determine concentrations (and prepare for ligation of GVP behind LAC/pBAD promoters)

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

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

→ DONE Transform E.coli TOP10 cells with [http://partsregistry.org/Part:pSB2K3 pSB2K3] plasmid from the registry (plate 1, 7C with RFP Coding Device [http://partsregistry.org/Part:BBa_J04450 BBa_J04450], and plate 1, 7K with ccdB cell death gene [http://partsregistry.org/Part:BBa_P1010 BBa_P1010])

→ TODO Test control of bouyancy in Saline solution

→ TODO Order synthetic DNA for GVP

→ TODO Order primer for PstI site removal

→ 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, the ones with 10mL LB were less dense as expected). To be sure of correct ligation products in the plasmids an isolation will be performed, followed by restriction analysis.

Overnight Plates

The plates grown overnight showed single colony growth, the plates were stored in the fridge for preculture inoculation.

Transformation

The [http://partsregistry.org/Part:pSB2K3 pSB2K3] vector was chosen to use together with the [http://partsregistry.org/Part:pSB1AC3 pSB1AC3] vector to create a two plasmid system. The ME^tal promoters with GVP on pSB2K3, and accumulation with transport proteins on pSB1AC3. These two vectors should be compatible with respect to their origins of replication.

• add 3uL 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.

Transporters

Metal Accumulation

MBP-ArsR

• Check sequence
• Put promotors in front
• Put transporters in back

fMT

• Plasmid isolation & Restriction analysis
• Put promotors in front

SmtA

• Plasmid isolation & Restriction analysis
• Put promotors in front

Vectors

Dry