Team:Groningen/Notebook/10 September 2009

From 2009.igem.org

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Wet

GVP Cluster

O.n. precultures

→ All tubes for plasmid isolation (pLacI (pSB1A2)(no.1 and no.2), pSB2K3 (12J)(2x) and pSB2K3 (12L)(2x) showed growth. The pSB2K3 had less growth (and smaller pellet) than the other tubes.


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Plasmid isolation

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 (tubes from pSB2K3 12J and 12L were combined), 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
pLacI no.1 (pSB1A2) 66.3 1.86 1.82 x Yes (SpeI/PstI)
pLacI no.2 (pSB1A2) 47.1 1.82 1.45 x Yes (SpeI/PstI)
pSB2K3 (12J) 142.9 1.89 2.31 x Yes (EcoRI/PstI)
pSB2K3 (12L) 337.1 1.87 2.35 x Yes (EcoRI/PstI)


Restriction for Assembly

The plasmids from the o.n. precultures were cut with PstI and EcoRI to cut out the entire part between the pre- and suffix.

Plasmid Amount μL MQ μL Fast digest buffer EcoRI fast digest enzyme XbaI fast digest enzyme SpeI fast digest enzyme PstI fast digest enzyme
pLacI no.1 (pSB1A2) 16.0 x 3.0 x x 1.0 1.0
pLacI no.1 (pSB1A2) 16.0 x 3.0 x x 1.0 1.0
GVP 4.0 11.0 3.0 x 1.0 x 1.0
pSB2K3 (12J) 8.0 6.0 3.0 1.0 x x 1.0
pSB2K3 (12L) 4.0 11.0 3.0 1.0 x x 1.0
pZntR-GVP 12.0 3.0 3.0 1.0 x x 1.0


10-9 no.1.jpg Generulers 1kb marker Fermentas.jpg

→ From left to right: 1kB ladder, pSB2K3 12J (2x), Empty Slot, pSB2K3 12L (2x), Empty Slot, pZntR-GVP (2x)

10-9 no.2.jpg Generulers 1kb marker Fermentas.jpg

→ From left to right: 1kB ladder, pLacI no.1 (pSB1A2)(2x), pLacI no.2 (pSB1A2)(2x), GVP (2x)


Purification

www.zymoresearch.com


→ In step 7 the fragments were eluted in 12μL MQ and was stored on ice until use.


Concentration


Ligation

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

  • 3 uL Ligase buffer
  • 1 ul T4 Ligase
  • uL plasmid pSB1A2 pLacI digested with SpeI and PstI
  • uL GVP restricted with XbaI and PstI


  • 3 uL Ligase buffer
  • 1 ul T4 Ligase
  • uL plasmid pSB2K3 digested with EcoRI and PstI
  • uL pZntR-GVP and pCueO-GVP restricted with EcoRI and PstI
  • uL MQ

Incubate:

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

Tranformation

  • add 10uL 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.


Observations

Here are the allignments of the expect

Transporters

Metal Accumulation

Alignment fMT

Allignments fMT.jpg

Alignments of the metallothionein fMT constructs, above is In silico construct, below is sequence determined with VF2 primer. The first is without promotor (BBa_K190019), second with constitutive promotor BBa_J23109 (BBa_K190031) and the third with IPTG inducible promotor BBa_R0010 (BBa_K190032). RBS is BBa_B0034. In combining the promotor and the coding sequence the Partregistry seems to add two unexpected additional bases (ag) between the two, missing in sequenced sample. In preparing the In silico construct a different prefix (lacking two bases (ag)), seen in sequenced samples, was used. The n's in the suffix of BBa_K190032 were determined to be a g as expected when giving a closer look into the chromatogram.

Alignment MBP-ArsR

Allignment MBP-ArsR.jpg

Alignments of the metal chelator Maltose Binding Protein (MBP)-ArsR fusion protein (BBa_K190027). Above is In silico construct, in the middle is sequence determined with VF2 primer, below the sequence determined with VR primer. A Tobacco Etch Virus (TEV) protease cleavage site is added between the two proteins.

Vectors

Dry

April
MTWTFSS
    1 2 3 4 5
6 7 8 9 10 11 12
13 14 15 16 17 18 19
20 21 22 23 24 25 26
27 28 29 30
May
MTWTFSS
        1 2 3
4 5 6 7 8 9 10
11 12 13 14 15 16 17
18 19 20 21 22 23 24
25 26 27 28 29 30 31
June
MTWTFSS
1 2 3 4 5 6 7
8 9 10 11 12 13 14
15 16 17 18 19 20 21
22 23 24 25 26 27 28
29 30
July
MTWTFSS
    1 2 3 4 5
6 7 8 9 10 11 12
13 14 15 16 17 18 19
20 21 22 23 24 25 26
27 28 29 30 31
August
MTWTFSS
          1 2
3 4 5 6 7 8 9
10 11 12 13 14 15 16
17 18 19 20 21 22 23
24 25 26 27 28 29 30
31
September
MTWTFSS
  1 2 3 4 5 6
7 8 9 10 11 12 13
14 15 16 17 18 19 20
21 22 23 24 25 26 27
28 29 30
October
MTWTFSS
      1 2 3 4
5 6 7 8 9 10 11
12 13 14 15 16 17 18
19 20 21 22 23 24 25
26 27 28 29 30 31
November
MTWTFSS
            1
2 3 4 5 6 7 8
9 10 11 12 13 14 15
16 17 18 19 20 21 22
23 24 25 26 27 28 29
30