Team:Groningen/Notebook/25 August 2009

From 2009.igem.org

(Difference between revisions)
(GVP Cluster)
(Metal promotors)
 
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:→ {{todo}} Gel purification of wanted fragments and nanodrop
:→ {{todo}} Gel purification of wanted fragments and nanodrop
:→ {{todo}} Clone GVP cluster behind Lac and Arabinose promoter for expression tests in pSB1AC3 plasmid
:→ {{todo}} Clone GVP cluster behind Lac and Arabinose promoter for expression tests in pSB1AC3 plasmid
-
:→ {{todo}} Send plasmids with ME<sup>tal</sup> promoters in BBa_J61002 vector for sequencing
+
:→ {{done}} Send plasmids with ME<sup>tal</sup> promoters in BBa_J61002 vector for sequencing
-
:→ {{todo}} Check ME<sup>tal</sup> + RBS promoters on gel with restriction (also XbaI/PstI in Zinc promoter)
+
:→ {{done}} Check ME<sup>tal</sup> + RBS promoters on gel with restriction (also XbaI/PstI in Zinc promoter)
:→ {{todo}} Test promoter strenght compared to BBa_J23101 promoter (Sven)
:→ {{todo}} Test promoter strenght compared to BBa_J23101 promoter (Sven)
-
:→ {{todo}} Enter sequences of constructs to Sandbox
+
:→ {{done}} Enter sequences of constructs to Sandbox
===Transporters===
===Transporters===
===Metal Accumulation===
===Metal Accumulation===
 +
*'''PCR to check MBP-Ars'''
 +
<center>[[Image:PCR_Check_MBP-ArsR_-_24august2009.jpg|250px]]</center><BR>
 +
PCR check of MPB-ArsR fusion protein, two combinations of primers were used; Forward MBP with Reverse ArsR & Forward ArsR with Reverse MBP, giving the indication where proteins are in comparison of each other. Only the first gave the expected product of 1500 bp, indicating that the fusion was as expected MBP-ArsR and nog ArsR-MBP. Sample was send for sequencing.
 +
 +
*'''Continue cloning SmtA and fMT in pSB1AC3 + promoters'''
 +
**Miniprep pSB1AC3-fMT and SmtA using the Sigma Kit.
 +
**Send the constructs for sequencing.
 +
**Digest pSB1AC3-fMT and SmtA with XbaI and PstI, digest pSB1AC3-pLow (), pSB1AC3-pLac and pSB1AC3-pBAD with PstI and SpeI.
 +
**Run the restriction products on a 1% Agarose gel
 +
[[Image:F102471 2009-08-25 fmt smta ac digest.JPG|250px]]
 +
::-From left to right: Marker, SmtA 2/X-P, SmtA 3/X-P, fMT 4/X-P, fMT 6/X-P, fMT 6/X-P(only a few uL), pSB1AC3-pLow/S-P, pSB1AC3-pLac/S-P, pSB1AC3-pBAD/S-P, marker.
 +
::-Expected size: SmtA 900bp, fMT 200bp, pSB1AC3 3300bp, pSB1AC3-pBAD 3500bp.
 +
**The bands of SmtA 2/X-P were different from SmtA 3/X-P, it seems like an extra restriction site has been inserted into the gene as the fragment sizes are ~600 + ~300.
 +
**The correct bands were excised and the DNA purified using the Roche Kit.
 +
**Ligation was done overnight @ 4&deg;C of pSB1AC3-pLow + fMT and SmtA and pSB1AC3-pLac + fMT and SmtA as the concentration of pSB1AC3-pBAD was way to low...
===Vectors===
===Vectors===
====Metal promotors====
====Metal promotors====
-
Cloning into pSB1AC3 and pSB3K3 has stopped
+
'''Cloning of the metal promoters into pSB1AC3 and pSB3K3 has stopped'''
==Dry==
==Dry==
 +
Jasper finally finished simplifying the differential equations and they are now presented in neat tables on [[Team:Groningen/Modelling/Arsenic|our modelling page]]. It turned out that it is easiest to work with differential equations for the '''totals''' of substances in combination with relative abundances of the different forms of the substances.
{{Team:Groningen/Notebook/Day/Footer}}
{{Team:Groningen/Notebook/Day/Footer}}

Latest revision as of 16:47, 25 August 2009

Igemhomelogo.png

Wet

GVP Cluster

TODO Restriction of GVP and pSB1AC3-Lac/pBAD for assembly
TODO Gel purification of wanted fragments and nanodrop
TODO Clone GVP cluster behind Lac and Arabinose promoter for expression tests in pSB1AC3 plasmid
DONE Send plasmids with MEtal promoters in BBa_J61002 vector for sequencing
DONE Check MEtal + RBS promoters on gel with restriction (also XbaI/PstI in Zinc promoter)
TODO Test promoter strenght compared to BBa_J23101 promoter (Sven)
DONE Enter sequences of constructs to Sandbox

Transporters

Metal Accumulation

  • PCR to check MBP-Ars
PCR Check MBP-ArsR - 24august2009.jpg

PCR check of MPB-ArsR fusion protein, two combinations of primers were used; Forward MBP with Reverse ArsR & Forward ArsR with Reverse MBP, giving the indication where proteins are in comparison of each other. Only the first gave the expected product of 1500 bp, indicating that the fusion was as expected MBP-ArsR and nog ArsR-MBP. Sample was send for sequencing.

  • Continue cloning SmtA and fMT in pSB1AC3 + promoters
    • Miniprep pSB1AC3-fMT and SmtA using the Sigma Kit.
    • Send the constructs for sequencing.
    • Digest pSB1AC3-fMT and SmtA with XbaI and PstI, digest pSB1AC3-pLow (), pSB1AC3-pLac and pSB1AC3-pBAD with PstI and SpeI.
    • Run the restriction products on a 1% Agarose gel

F102471 2009-08-25 fmt smta ac digest.JPG

-From left to right: Marker, SmtA 2/X-P, SmtA 3/X-P, fMT 4/X-P, fMT 6/X-P, fMT 6/X-P(only a few uL), pSB1AC3-pLow/S-P, pSB1AC3-pLac/S-P, pSB1AC3-pBAD/S-P, marker.
-Expected size: SmtA 900bp, fMT 200bp, pSB1AC3 3300bp, pSB1AC3-pBAD 3500bp.
    • The bands of SmtA 2/X-P were different from SmtA 3/X-P, it seems like an extra restriction site has been inserted into the gene as the fragment sizes are ~600 + ~300.
    • The correct bands were excised and the DNA purified using the Roche Kit.
    • Ligation was done overnight @ 4°C of pSB1AC3-pLow + fMT and SmtA and pSB1AC3-pLac + fMT and SmtA as the concentration of pSB1AC3-pBAD was way to low...

Vectors

Metal promotors

Cloning of the metal promoters into pSB1AC3 and pSB3K3 has stopped

Dry

Jasper finally finished simplifying the differential equations and they are now presented in neat tables on our modelling page. It turned out that it is easiest to work with differential equations for the totals of substances in combination with relative abundances of the different forms of the substances.


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