Team:Groningen/Notebook/28 August 2009

From 2009.igem.org

(Difference between revisions)
(Transporters)
m (GVP Cluster)
Line 25: Line 25:
:→ The colonies for pArsR+RBS showed red colonies, and the pCueO+RBS/pZntR+RBS showed normal yellow/white colonies.
:→ The colonies for pArsR+RBS showed red colonies, and the pCueO+RBS/pZntR+RBS showed normal yellow/white colonies.
-
'''Restriction for Assembly'''
+
'''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.  
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.  
Line 37: Line 37:
Restriction was kept at 37C for 30 min., followed by addition of 6x loading dye and put on ice until used for gel analysis.
Restriction was kept at 37C for 30 min., followed by addition of 6x loading dye and put on ice until used for gel analysis.
 +
[[Image:28-8 no.1.jpg|350px]] [[Image:Generulers_1kb_marker_Fermentas.jpg]]
 +
 +
:→ 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....
:→ [http://partsregistry.org/Part:BBa_R0010 pLacI]
:→ [http://partsregistry.org/Part:BBa_R0010 pLacI]

Revision as of 13:36, 28 August 2009

Igemhomelogo.png

Wet

GVP Cluster

DONE Isolate plasmids from o.n. cultures
TODO 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 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.

28-8 no.1.jpg Generulers 1kb marker Fermentas.jpg

→ 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....
pLacI
pBad/araC
pSB1A2


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.

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.

Metal Accumulation

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