Team:Groningen/Notebook/29 July 2009

From 2009.igem.org

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(Dry log.)
(Vectors)
 
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===Transporters===
===Transporters===
 +
GlpF
 +
Transformation of GlpF in pSB1AC3 + medium constitutive promotor, pNL29 and SmtA
 +
{|
 +
|DNA
 +
|Amount added to competent cells
 +
|antibiotic
 +
|-
 +
|Ligation mix GlpF psB1AC3-M
 +
|4 uL
 +
|Amp
 +
|-
 +
|Cut psB1AC3+M
 +
|2 uL
 +
|Amp
 +
|-
 +
|Neg controle
 +
|0 uL
 +
|geen
 +
|-
 +
|pNL29 (Gvp)
 +
|0.5uL
 +
|Amp
 +
|-
 +
|SmtA GST tagged
 +
|2 uL
 +
|Amp
 +
|-
 +
|SmtA untagged
 +
|2 uL
 +
|Kan
 +
|-
 +
|}
 +
 +
 +
The cells were incubated (30 min, on ice; 5 min 37; 5 min on ice)
 +
800uLm LB was added
 +
The cells were incubated for 1h at 37
 +
The cells wer plated on and incubated at 37 overnight.
 +
No colonies were seen on the GlpF-psB1AC3, psB1AC3 resticted, pNL and the negative control. Colonies were seen on the SmtA tagged and untagged plates.
===Metal Accumulation===
===Metal Accumulation===
===Vectors===
===Vectors===
 +
Ligation reactions were transformed into 50 μL TOP10 cells using heatshock, 45 sec. 42 °C. Cells were left to recover for 1 h at 37 °C after adding 200 μL LB medium. Cell suspensions plated out along with a negative control (1 μL MilliQ water) and a positive control (1 μL) {{part|pSB1AC3}}-High or {{part|pSB3K3}}-H. Samples were plated out in volumes of 50 μL and 200 μL and incubated ON at 37 °C.
 +
 +
 +
*'''Restriction digest to ligate vectors+constitutive promoters with the RFP device''' and '''pBAD and pLac with pSB3K3/pSB1AC3'''
 +
 +
**Isolate vector+ pBAD promoter with Sigma Plasmid isolation kit.
 +
**Restriction digest as following:
 +
 +
{|
 +
|
 +
<!--Tabel 1 hier-->
 +
|width="10"|
 +
|
 +
 +
{| border="1"
 +
'''Restriction digest on pLac/pBAD'''
 +
 +
|10x FD buffer
 +
|2 uL
 +
|-
 +
|pSB1A2-pLac/pBAD
 +
|17 uL
 +
|-
 +
|EcoRI
 +
|1 uL
 +
|-
 +
|SpeI
 +
|1 uL
 +
|-
 +
|Total
 +
|20 uL
 +
|-
 +
|}
 +
 +
<!--Tabel 2 hier-->
 +
|width="10"|
 +
|
 +
 +
{| border="1"
 +
'''Restriction digest on pSB1AC3-const. promoter, pSB3K3-const. promoter'''
 +
 +
|10x FD buffer
 +
|2 uL
 +
|-
 +
|pSB3K3-HML or pSB1AC3-HML
 +
|13 or 17 uL (depending on conc.)
 +
|-
 +
|SpeI
 +
|1 uL
 +
|-
 +
|PstI
 +
|1 uL
 +
|-
 +
|MilliQ
 +
|4 uL
 +
|-
 +
|Total
 +
|20 uL
 +
|-
 +
|}
 +
 +
<!--Tabel 3 hier-->
 +
|width="10"|
 +
|
 +
 +
{| border="1"
 +
'''Restriction digest on pSB1A2-RBS-RFP'''
 +
 +
|10x FD buffer
 +
|2 uL
 +
|-
 +
|pSB1A2-RBS-RFP
 +
|4 uL
 +
|-
 +
|XbaI
 +
|1 uL
 +
|-
 +
|PstI
 +
|1 uL
 +
|-
 +
|MilliQ
 +
|12 uL
 +
|-
 +
|Total
 +
|20 uL
 +
|-
 +
|}
 +
 +
|}
 +
**Expected sizes of wanted fragments:
 +
***pBAD: 1700bp
 +
***pLac: 200bp
 +
***pSB3K3-const. promoter: 3000
 +
***pSB1AC3-const. promoter: 2700
 +
***RBS-RFP: 1000bp
 +
**Cut bands from gel (pSB1AC3-M showed no bands at all, so nothing to cut out..)
 +
**DNA gel extraction with NucleoSpin Extract II
 +
**Elute with 15uL NE buffer.
 +
**DNA concentration was measured by NanoDrop, they were in the range of 35-5ng/uL.
 +
**Stored in the freezer (should be fridge)
==Dry==
==Dry==
Jasper made sure the graphing functionality can make use of tables on the Wiki. It now can (see the [[Team:Groningen/Graphs/TestPie|pie chart test]] for an example). By default it takes the first table on the page, but if you append '#myid' it should use the table with id myid.
Jasper made sure the graphing functionality can make use of tables on the Wiki. It now can (see the [[Team:Groningen/Graphs/TestPie|pie chart test]] for an example). By default it takes the first table on the page, but if you append '#myid' it should use the table with id myid.
 +
 +
Also, since the information on some of the other candidates to model doesn't seem to be sufficient and the lab work is still more or less a moving target anyway (and in fact ArsR and GlpF might even be "ahead" in the cloning race right now) we decided to stick to our arsenic model(s). So instead of focussing on new models we're going to improve the existing ones, have a look at the underlying technology (like the wiki table functionality mentioned above) and think about experiments.
 +
 +
Working to the model accumulation MT in matlab [[Team:Groningen/Literature#Ngu2006|Ngu2006]].
 +
Update of the literature table on the wiki, there is now a summery of the papers which are used for the metals.
 +
I want the text headers rotated, but its works only in chorme and mozzila.
{{Team:Groningen/Notebook/Day/Footer}}
{{Team:Groningen/Notebook/Day/Footer}}

Latest revision as of 15:00, 3 August 2009

Igemhomelogo.png

Wet

GVP Cluster

Transporters

GlpF Transformation of GlpF in pSB1AC3 + medium constitutive promotor, pNL29 and SmtA

DNA Amount added to competent cells antibiotic
Ligation mix GlpF psB1AC3-M 4 uL Amp
Cut psB1AC3+M 2 uL Amp
Neg controle 0 uL geen
pNL29 (Gvp) 0.5uL Amp
SmtA GST tagged 2 uL Amp
SmtA untagged 2 uL Kan


The cells were incubated (30 min, on ice; 5 min 37; 5 min on ice) 800uLm LB was added The cells were incubated for 1h at 37 The cells wer plated on and incubated at 37 overnight. No colonies were seen on the GlpF-psB1AC3, psB1AC3 resticted, pNL and the negative control. Colonies were seen on the SmtA tagged and untagged plates.

Metal Accumulation

Vectors

Ligation reactions were transformed into 50 μL TOP10 cells using heatshock, 45 sec. 42 °C. Cells were left to recover for 1 h at 37 °C after adding 200 μL LB medium. Cell suspensions plated out along with a negative control (1 μL MilliQ water) and a positive control (1 μL) pSB1AC3-High or pSB3K3-H. Samples were plated out in volumes of 50 μL and 200 μL and incubated ON at 37 °C.


  • Restriction digest to ligate vectors+constitutive promoters with the RFP device and pBAD and pLac with pSB3K3/pSB1AC3
    • Isolate vector+ pBAD promoter with Sigma Plasmid isolation kit.
    • Restriction digest as following:
Restriction digest on pLac/pBAD
10x FD buffer 2 uL
pSB1A2-pLac/pBAD 17 uL
EcoRI 1 uL
SpeI 1 uL
Total 20 uL
Restriction digest on pSB1AC3-const. promoter, pSB3K3-const. promoter
10x FD buffer 2 uL
pSB3K3-HML or pSB1AC3-HML 13 or 17 uL (depending on conc.)
SpeI 1 uL
PstI 1 uL
MilliQ 4 uL
Total 20 uL
Restriction digest on pSB1A2-RBS-RFP
10x FD buffer 2 uL
pSB1A2-RBS-RFP 4 uL
XbaI 1 uL
PstI 1 uL
MilliQ 12 uL
Total 20 uL
    • Expected sizes of wanted fragments:
      • pBAD: 1700bp
      • pLac: 200bp
      • pSB3K3-const. promoter: 3000
      • pSB1AC3-const. promoter: 2700
      • RBS-RFP: 1000bp
    • Cut bands from gel (pSB1AC3-M showed no bands at all, so nothing to cut out..)
    • DNA gel extraction with NucleoSpin Extract II
    • Elute with 15uL NE buffer.
    • DNA concentration was measured by NanoDrop, they were in the range of 35-5ng/uL.
    • Stored in the freezer (should be fridge)

Dry

Jasper made sure the graphing functionality can make use of tables on the Wiki. It now can (see the pie chart test for an example). By default it takes the first table on the page, but if you append '#myid' it should use the table with id myid.

Also, since the information on some of the other candidates to model doesn't seem to be sufficient and the lab work is still more or less a moving target anyway (and in fact ArsR and GlpF might even be "ahead" in the cloning race right now) we decided to stick to our arsenic model(s). So instead of focussing on new models we're going to improve the existing ones, have a look at the underlying technology (like the wiki table functionality mentioned above) and think about experiments.

Working to the model accumulation MT in matlab Ngu2006. Update of the literature table on the wiki, there is now a summery of the papers which are used for the metals. I want the text headers rotated, but its works only in chorme and mozzila.


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