Team:Groningen/Notebook/4 September 2009

From 2009.igem.org

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Wet

GVP Cluster

BBa_F2620

DONE Isolate plasmid from o.n. precultures of pLacI-GVP and pBad-araC-GVP in pSB1A2
DONE Restriction control of plasmids and purification of wanted fragments for ligation in pSB2K3 vector
DONE Ligate pArsR-GVP, pZntR-GVP, pCueO-GVP, and pLacI-GVP into vector pSB2A3
DONE Transform E.coli Top10 cells with ligation products and pSB2K3 (7C and 7K)
DONE Order synthetic DNA for GVP
TODO Order primer for PstI site removal
DONE Test promoter strenght compared to BBa_J23101 promoter (Sven)
TODO Enter sequences of constructs to Sandbox

O.n. precultures

→ All four o.n. precultures showed bacterial growth, and could be used to isolate plasmid. The four are registry vector pSB1A2 with pLacI/pBad-araC and GVP. The isolated plasmid is used for transformation into pSB2K3 and sent for sequencing.


www.sigmaaldrich.com

Plasmid Purification

Plasmid isolation was performed on the cultures of E.coli TOP10 containing the above mentioned plasmids 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


Concentrations

Plasmid Conc. ng/μL 260/280 260/230 -20 box (michael Restriction Control
pSB1A2 pLacI-GVP no.1 405.3 1.85 2.36 D-6 Yes (EcoRI/PstI)
pSB1A2 pLacI-GVP no.2 34.0 2.01 2.20 D-7 Yes (EcoRI/PstI)
pSB1A2 pBad/araC-GVP no.1 27.5 2.09 2.16 D-8 Yes (EcoRI/PstI)
pSB1A2 pBad/araC-GVP no.2 33.3 1.96 2.06 D-9 Yes (EcoRI/PstI)


Restriction for Assembly

The vector pSB1A2 containing the pLacI and pBad-araC with GVP composite parts were cut with PstI and EcoRI to create correct ends for insert into pSB2K3, which was also cut with EcoRI and PstI (4x) on 3-9.

Plasmid Amount μL MQ μL Fast digest buffer EcoRI fast digest enzyme XbaI fast digest enzyme SpeI fast digest enzyme PstI fast digest enzyme
pSB1A2 pLacI-GVP no.1 3.0 13.0 3.0 1.0 x x 1.0
pSB1A2 pLacI-GVP no.2 16.0 x 3.0 1.0 x x 1.0
pSB1A2 pBad/araC-GVP no.1 16.0 x 3.0 1.0 x x 1.0
pSB1A2 pBad/araC-GVP no.2 16.0 x 3.0 1.0 x x 1.0

Restriction was kept at 37C for 40 min. and put on ice until used for gel purification.


www.zymoresearch.com


Gel Purification

  • In step 7 an amount of 10μL MQ was added to elute the DNA fragments.


4-9 no.1.jpg Generulers 1kb marker Fermentas.jpg

→ Only the first restriction of pLacI-GVP seems to have worked judging from the fragments.

Ligation

A total amount of vector of 100ng was used (GVP) in a 1:3 ratio with insert.

Ligase buffer (uL) T4 Ligase (uL) Plasmid Insert MQ
3.0 1.0 5.0 pSB2K3 (no.2)(EcoRI,PstI) 3.0 pLacI-GVP (EcoRI,PstI) 8.0
3.0 1.0 5.0 pSB2K3 (no.2)(EcoRI,PstI) 8.0 pArsR-GVP (EcoRI,PstI) 3.0
3.0 1.0 6.0 pSB2K3 (no.3)(EcoRI,PstI) 8.0 pZntR-GVP (EcoRI,PstI) 2.0
3.0 1.0 6.0 pSB2K3 (no.3)(EcoRI,PstI) 8.0 pCueO-GVP (EcoRI,PstI) 2.0


Incubate:

  • 25°C 50min.
  • kept on ice for 10min.

Tranformation

Together with these four ligation products, the plasmids from plate 1, 7C and 7K were transformed and grown on LB-kan50-IPTG plates to see if a higher colony number could be reached.

  • add 10uL of the ligation product to 50uL competent E.coli TOP10 cells.
  • add 2uL of the 7C and 7K plasmids to 50uL competent E.coli TOP10 cells.

Incubate:

  • 30 min @ ice
  • 90 sec 42°C
  • 2 min @ ice
  • add 800uL LB-medium-IPTG
  • incubate for 1 h at 37°C
  • plate on LB-kan50-IPTG plates

phase contrast microscope

The three pictures below show 1000X magnification of pNL29induced for one day (1) for a week(2) and non induced (3)

In the first picture we see some light cell contend compared tot non induced cells. The second picture of an old culture we believe we see the gas vesicles clustered in the bright spot.

Transporters

Metal Accumulation

Made a SmtA-1 ON culture, the assumption that the fragment should have been ~1200 bp was false and the sequencing results showed that neither SmtA-2 or SmtA-3 contained the proper construct. BBa_K190021 should be around ~700 bp including prefix and suffix, instead of 900 bp. As SmtA-1 was positioned lower than the others it might contain the proper construct.

Glycerol stocks (-80 °C)

Component Description Part or Accession # Base Pairs (bp) Plasmid (backbone) Resistance Well Quality control Storage Medium+Antibiotics Organism Date of storage
fMT #4 Metallothionein for As3+ BBa_K190019 222 pSB1AC3 Ampicillin/chloramphenicol plate 3, pos. 60 Sequence Okay! LB-Ampicillin E. coli TOP10 04september2009

Vectors

Dry

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