Team:Groningen/Notebook/4 August 2009

From 2009.igem.org

(Difference between revisions)
(Dry)
(Transporters)
 
(12 intermediate revisions not shown)
Line 111: Line 111:
===Transporters===
===Transporters===
-
GlpF  
+
'''GlpF'''
Restriction, ligation and transformation. Done as noted at [https://2009.igem.org/Team:Groningen/Notebook/31_July_2009| 31 July]
Restriction, ligation and transformation. Done as noted at [https://2009.igem.org/Team:Groningen/Notebook/31_July_2009| 31 July]
-
.
+
. only the inactivation step was not done using a agarose gel but by using a PCR clean up kit (Sigma-Aldrich).
===Metal Accumulation===
===Metal Accumulation===
-
Fusion ArsR with MBP
+
'''Fusion ArsR with MBP'''
-
Restriction
+
''Restriction''
{|
{|
|
|
{|
{|
|10ul
|10ul
-
|GlpF PCR
+
|MBP
|-
|-
|2 ul
|2 ul
-
|Buffer
+
|FastDigestBuffer
|-
|-
-
|05.ul
+
|1uL
-
|EcoRI
+
|SacI
|-
|-
-
|0.5 ul
+
|3 uL
-
|SpeI
+
-
|-
+
-
|7 uL
+
|MQ
|MQ
|-
|-
Line 141: Line 138:
|width="10"|
|width="10"|
|
|
-
<!--Tabel 2 hier-->
+
 
 +
{|
 +
|10ul
 +
|ArsR
 +
|-
 +
|2 ul
 +
|FastDigestBuffer
 +
|-
 +
|0.5ul
 +
|SacI
 +
|-
 +
|1.5 uL
 +
|MQ
 +
|-
 +
|}
|width="10"|
|width="10"|
|
|
<!--Tabel 3 hier-->
<!--Tabel 3 hier-->
|}
|}
-
: '''Restriction'''
+
 
 +
:*incubated 37&deg; 30 min
 +
:*inactivated enzyme at 65&deg; for 5 min
 +
 
 +
''Ligation''
{|
{|
 +
|1 uL
 +
|T4 Ligase
 +
|-
 +
|2 ul
 +
|T4 Ligase Buffer
 +
|-
 +
|10 ul
 +
|digested MBP
 +
|-
 +
|3 uL
 +
|digested ArsR
 +
|-
 +
|4 uL
 +
|MQ
 +
|}
 +
:*incubated at 4&deg; overnight
===Vectors===
===Vectors===
Line 153: Line 184:
==Dry==
==Dry==
Jasper did some "maintenance" on the Wiki and created a diagram of the processes involved in arsenic filtering.
Jasper did some "maintenance" on the Wiki and created a diagram of the processes involved in arsenic filtering.
 +
Kb made the final changes to the brochure. This is the final version
Kb made the final changes to the brochure. This is the final version
 +
 +
The ‘dojo’ graph doesn’t work in Mozzila firefoxs. This problem is fixed trough set both the id and name attributes of the iframe.  http://www.dyn-web.com/tutorials/iframes/
 +
{{Team:Groningen/Notebook/Day/Footer}}
{{Team:Groningen/Notebook/Day/Footer}}

Latest revision as of 15:18, 4 August 2009

Igemhomelogo.png

Wet

GVP Cluster

All precultures containing a plasmid with a promoter have grown to the expected density! Time for some plasmid isolation to be carried out...

DONE weekly meeting (9 to 12)
DONE isolate plasmids from precultures
DONE perform a restriction on the medium promoter containing plasmids with SpeI and PstI
DONE purify cut vectors with miniprep (11bp fragment should be lost)
TODO perform a ligation between cut vector and GVP fragment
TODO transform E.coli TOP10 competent cells with ligation product


www.sigmaaldrich.com

Plasmid Purification

Plasmid isolation was performed on the cultures of E.coli TOP10 containing plasmids pSB3K3 and pSB1AC3 with high, medium and low constitutive promoters with the "Sygma-Aldrich™ GenElute™ Plasmid Miniprep Kit".

  • From each tube 3mL of culture was collected in a 1.5mL cup, and the cells were pelleted by centrifugation for 1 min. at max. speed and the supernatant discarded.
  • Plasmids were eluted with 50μL MQ and stored in the fridge

Concentration

The concentration of isolated plasmid was determined with the use of a nano-drop.

pSB3K3-highP in MQ

  • 11.3 ng/μL
  • 0.227 (260/280)
  • 0.114 (260/230)

pSB3K3-mediumP in MQ

  • 17.2 ng/μL
  • 0.344 (260/280)
  • 0.201 (260/230)

pSB3K3-lowP in MQ

  • 12.9 ng/μL
  • 0.257 (260/280)
  • 0.143 (260/230)

pSB1AC3-highP in MQ

  • 114.0 ng/μL
  • 2.279 (260/280)
  • 1.335 (260/230)

pSB1AC3-mediumP in MQ

  • 55.9 ng/μL
  • 1.118 (260/280)
  • 0.614 (260/230)

pSB1AC3-lowP in MQ

  • 47.9 ng/μL
  • 0.957 (260/280)
  • 0.512 (260/230)

Restriction for Assembly

The vector pSB1AC3 containing the high, medium and low constitutive promoters were cut with PstI and SpeI to create correct ends for insert of GVP biobrick BBa_I750016.

  • 16μL plasmid in MQ (0.8-1.6μg)
  • 2μL Fast digest buffer
  • 1μL PstI fast digest enzyme
  • 1μL SpeI fast digest enzyme

Vector Purification

Cut vector purification was performed on the restricted plasmid pSB1AC3 containing the high, medium and low constitutive promoters with the "Sygma-Aldrich™ GenElute™ Plasmid Miniprep Kit". The 11bp fragment between the SpeI and PstI restriction sites should be lost in this way, and gel purification may be omitted.

  • After adding 200μL of resuspention buffer, 200μL lysis buffer and 350μL neutralization buffer the solution was centrifuged for 10min. and the standard protocol was followed.
  • Cut vectors were eluted with 20μL MQ and stored in the fridge

Concentration

The concentration of purified cut vector was determined with the use of a nano-drop.

pSB1AC3-highP-res. in MQ

  • 14.9 ng/μL
  • 0.278 (260/280)
  • 0.172 (260/230)

pSB1AC3-mediumP-res. in MQ

  • 19.7 ng/μL
  • 0.394 (260/280)
  • 0.204 (260/230)

pSB1AC3-lowP-res. in MQ

  • 17.5 ng/μL
  • 0.349 (260/280)
  • 0.210 (260/230)

Over Night Cultures

Over night cultures in 4mL LB-amp100 medium were prepared from the remaining overnight cultures of:

→ E.coli TOP10 pSB3K3-high const. promoter (kan.)
→ E.coli TOP10 pSB3K3-med. const. promoter (kan.)
→ E.coli TOP10 pSB3K3-low const. promoter (kan.)

and put in the 37°C waterbath at 200 rpm. This was done because the low copy plasmid resulted in a to low isolated concentration. Longer growth and elution in less MQ should increase the recovered amount.


Over night culture in 5mL LB-amp100 medium was prepared from the following glycerol stock:

→ E.coli TOP10 GVP1 (amp.)

and put in the 37°C waterbath at 200 rpm.

Transporters

GlpF

Restriction, ligation and transformation. Done as noted at 31 July . only the inactivation step was not done using a agarose gel but by using a PCR clean up kit (Sigma-Aldrich).

Metal Accumulation

Fusion ArsR with MBP

Restriction

10ul MBP
2 ul FastDigestBuffer
1uL SacI
3 uL MQ
10ul ArsR
2 ul FastDigestBuffer
0.5ul SacI
1.5 uL MQ
  • incubated 37° 30 min
  • inactivated enzyme at 65° for 5 min

Ligation

1 uL T4 Ligase
2 ul T4 Ligase Buffer
10 ul digested MBP
3 uL digested ArsR
4 uL MQ
  • incubated at 4° overnight

Vectors

Dry

Jasper did some "maintenance" on the Wiki and created a diagram of the processes involved in arsenic filtering.

Kb made the final changes to the brochure. This is the final version

The ‘dojo’ graph doesn’t work in Mozzila firefoxs. This problem is fixed trough set both the id and name attributes of the iframe. http://www.dyn-web.com/tutorials/iframes/


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