Team:Freiburg/Notebook/12 August

From 2011.igem.org

(Difference between revisions)
(→Lysis cassette)
 
(19 intermediate revisions not shown)
Line 1: Line 1:
{{:Team:Freiburg/Templates/header}}
{{:Team:Freiburg/Templates/header}}
 +
<html>
 +
<div id="notebook-page-header">
 +
<div id="notebook-back" width="100px" >
 +
<a href="https://2011.igem.org/Team:Freiburg/Notebook/11_August">Previous entry</a>
 +
</div>
 +
<div id="notebook-title">
 +
<a href="https://2011.igem.org/Team:Freiburg/Notebook"> 12 August </a>
 +
</div>
 +
<div id="notebook-next">
 +
<a href="https://2011.igem.org/Team:Freiburg/Notebook/13_August">Next entry</a>
 +
</div>
 +
</div>
 +
</html>
 +
==Meeting==
==Meeting==
-
attendants: <br/>
+
attendants: Jakob, Julia, Manuel, Sandra, Sophie, Theo, Tobias  <br/>
-
Time:  
+
Time: 10:00 - 11:30
===<span style="color:green;">green light receptor</span>===
===<span style="color:green;">green light receptor</span>===
already done:
already done:
-
 
+
*quick change with CcaR didn't work
 +
*colonies for pcyA and ho1
To-do:
To-do:
 +
*order new primer for the quick change
 +
*cph8: we will use the primer and protocol from the Upsalla team
 +
*test if the colonies are positive
===<span style="color:blue;">blue light receptor</span>===
===<span style="color:blue;">blue light receptor</span>===
Line 17: Line 35:
already done:  
already done:  
-
 
+
*PCR to amplifiy the receptor with new primer pair
To-do:  
To-do:  
 +
*check if the PCR worked
===<span style="color:red;">red light receptor</span>===
===<span style="color:red;">red light receptor</span>===
already done:
already done:
-
 
To-do:
To-do:
Line 31: Line 49:
already done:
already done:
-
 
+
*lysis cassette with RBS
 +
*colonies for the GFP-PBD
 +
*inducible promotor + PR done
To-do:
To-do:
-
 
+
*overnight culture of the GFP-PDB colonies, miniprep, test digest and sequencing
===<span style="color:grey;">Precipitator</span>===
===<span style="color:grey;">Precipitator</span>===
already done:
already done:
-
 
+
*waiting for the genes
To-do:
To-do:
 +
still more waiting to do
 +
 +
===other stuff===
 +
*only some teams/people replied to the code we sent around
 +
*Rüdiger will meet Hauke Busch who will help us along with the modelling
 +
*everyone should do a sketch for his iGEM parts and constructs (also a short text to explain the function of every part)
 +
*the skype talk to the Upsalla team was helpful: we exchanged the primer sequences and some further experiences we had with the parts and with iGEM itself
 +
*we still need the official OK that we can collect donation about the crowd funding
 +
*Tobi wants to contact some newspapers
 +
*we want to practice our talk in front of the MPI people and the bioss people, we should get appointments, Sandra wants to ask Miriam from the MPI if we could do the talk on friday 23rd or wednesday 28th
 +
*we still need to update the wiki gallery
 +
<br/>
<br/>
<br/>
<br/>
 +
==<span style="color:green;">green light receptor</span>==
==<span style="color:green;">green light receptor</span>==
Line 59: Line 92:
'''Investigators: Sandra'''
'''Investigators: Sandra'''
-
To confrim the PCR worked this time, we loaded the sample onto a gel. The size of the fragment should be 3000bp.
+
To confirm the PCR worked this time, we loaded the sample onto a gel. The size of the fragment should be 1000bp.
 +
The gel showed a band at 1000bp.
 +
We digested the PCR product with DpnI and then purified the DNA with the PCR purification kit to further use the PCR product for 3A-assembly.
 +
 
 +
===3A-assembly of Not-Gate, LovTAP in pSB1T3===
 +
 
 +
'''Investigators: Sandra'''
 +
 
 +
'''Digestion'''
 +
 
 +
Amount of DNA and H20:
 +
 
 +
{| cellpadding="10" cellspacing="0" border="1"
 +
|Sample
 +
|DNA μl
 +
|H20 μl
 +
|-
 +
|LovTAP
 +
|5
 +
|33
 +
|-
 +
|Not-Gate
 +
|2
 +
|36
 +
|-
 +
|pSB1T3
 +
|20
 +
|18
 +
|}
 +
 
 +
 
 +
Enzymes necessary for digestion:
 +
 
 +
{|cellpadding="10" cellspacing="0" border="1"
 +
|
 +
|LovTAP
 +
|Not-Gate
 +
|vector
 +
|-
 +
|enzyme 1
 +
|EcoRI
 +
|XbaI
 +
|EcoRI
 +
|-
 +
|enzyme 2
 +
|NheI
 +
|PstI
 +
|PstI
 +
|}
 +
 
 +
 
 +
*Incubation at 37°C for 6 hours
 +
*20 minutes at 80°C
==<span style="color:red;">red light receptor</span>==
==<span style="color:red;">red light receptor</span>==
Line 106: Line 191:
-
{| style="border-spacing:0;"
 
-
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 
{| style="border-spacing:0;"
{| style="border-spacing:0;"
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Sample ID
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Sample ID
Line 278: Line 361:
|}
|}
-
 
+
<br>
-
|}
+
==<span style="color:grey;">Precipitator</span>==
==<span style="color:grey;">Precipitator</span>==
-
===NAME OF YOUR EXPERIMENT===
+
===Cloning===
 +
 
 +
'''Investigators: Sophie'''<br/>
 +
continue from experiment: Miniprep (Jakob/Ruediger, 12.8., see lysis cassette)<br/>
 +
project name: inducible promoter for Pbd<br/>
 +
parts: S54 (Eco & Spe)<br/>
 +
GFP-Pbd ε5 (Xba 7 Pst)<br/>
 +
psb1t3 (Eco & Pst)<br/>
 +
stored in: Ruediger's box<br/>
 +
 
 +
===Ligation===
 +
 
 +
'''Investigators: Sophie'''<br/>
 +
continue from experiment: Cloning (today, me)<br/>
 +
project name: inducible promoter for Pbd<br/>
 +
parts: S54 (Eco & Spe)<br/>
 +
GFP-Pbd ε5 (Xba 7 Pst)<br/>
 +
psb1t3 (Eco & Pst)<br/>
 +
name of ligation product: IPTG+Pbd<br/>
 +
stored in: Ruediger's box
 +
 
 +
===Testdigest===
-
'''Investigators: NAME'''
+
'''Investigators: Sophie'''<br/>
 +
continue from experiment: Ligation (today, me)<br/>
 +
project name: inducible promoter for Pbd<br/>
 +
[[File:Freiburg 12.8.11.jpg]]
 +
PR6 6 and Pr 4 4 were sequenced but PRs were missing again. Inducible promoter seems to be necessary.
{{:Team:Freiburg/Templates/footer}}
{{:Team:Freiburg/Templates/footer}}

Latest revision as of 01:07, 22 September 2011


This is the wiki page
of the Freiburger student
team competing for iGEM 2011.
Thank you for your interest!


Contents

Meeting

attendants: Jakob, Julia, Manuel, Sandra, Sophie, Theo, Tobias
Time: 10:00 - 11:30

green light receptor

already done:

  • quick change with CcaR didn't work
  • colonies for pcyA and ho1

To-do:

  • order new primer for the quick change
  • cph8: we will use the primer and protocol from the Upsalla team
  • test if the colonies are positive

blue light receptor

already done:

  • PCR to amplifiy the receptor with new primer pair

To-do:

  • check if the PCR worked

red light receptor

already done:

To-do:

Lysis cassette

already done:

  • lysis cassette with RBS
  • colonies for the GFP-PBD
  • inducible promotor + PR done

To-do:

  • overnight culture of the GFP-PDB colonies, miniprep, test digest and sequencing

Precipitator

already done:

  • waiting for the genes


To-do: still more waiting to do

other stuff

  • only some teams/people replied to the code we sent around
  • Rüdiger will meet Hauke Busch who will help us along with the modelling
  • everyone should do a sketch for his iGEM parts and constructs (also a short text to explain the function of every part)
  • the skype talk to the Upsalla team was helpful: we exchanged the primer sequences and some further experiences we had with the parts and with iGEM itself
  • we still need the official OK that we can collect donation about the crowd funding
  • Tobi wants to contact some newspapers
  • we want to practice our talk in front of the MPI people and the bioss people, we should get appointments, Sandra wants to ask Miriam from the MPI if we could do the talk on friday 23rd or wednesday 28th
  • we still need to update the wiki gallery



green light receptor

NAME OF YOUR EXPERIMENT

Investigators:NAME


blue light receptor

Gel of the PCR product: LovTAP

Investigators: Sandra

To confirm the PCR worked this time, we loaded the sample onto a gel. The size of the fragment should be 1000bp. The gel showed a band at 1000bp. We digested the PCR product with DpnI and then purified the DNA with the PCR purification kit to further use the PCR product for 3A-assembly.

3A-assembly of Not-Gate, LovTAP in pSB1T3

Investigators: Sandra

Digestion

Amount of DNA and H20:

Sample DNA μl H20 μl
LovTAP 5 33
Not-Gate 2 36
pSB1T3 20 18


Enzymes necessary for digestion:

LovTAP Not-Gate vector
enzyme 1 EcoRI XbaI EcoRI
enzyme 2 NheI PstI PstI


  • Incubation at 37°C for 6 hours
  • 20 minutes at 80°C

red light receptor

Picking clones of pcyA and ho1

Investigators: Sandra

Picked 2 clones of each plate:

  • 1pTd (PR1+pcyA+terminator)
  • 2pTd (PR2+pcyA+terminator)
  • 1pTb (PR1+pcyA+terminator)
  • 2pTb (PR2+pcyA+terminator)
  • 1L23 (PR1+ho1+terminator)
  • 2L23 (PR2+ho1+terminator)

Incubation over night.

Lysis cassette

Miniprep

Investigators: Jakob, Ruediger, Theo


Name: Jakob, Ruediger, Theo Date: 12.08.2011
Continue from Experiment: Transformation, 10.08.2011
Project Name:

Lysis systeme, plasticbinding, GST-Tag, IPTG inducible promotor, LOV-Tap

Documentation:

Why are you doing this experiment? Name the parts which you extract.

  • Need the DNA concentration

Describe your results and mistakes and measure the DNA concentration with the Nanodrop and note the results. How did you label your probes and where are they stored?


Sample ID Nuclein Acid Conc. ng/µl
A260/280
A260/230
LOV 3A 81,8 1,88 2,23
PR4+GFP 87,1 1,85 1,93
PR6-1 124,7 1,89 2,19
PR6-2 138,1 1,88 2,14
PR6-3 67,2 1,94 2,09
PR6-4 96,5 1,85 1,58
PR6-5 102,7 1,94 2,14
PR6-6 111,5 1,88 1,99
PR6-7 40,1 1,99 1,94
PR4-1 71,3 1,90 1,79
PR4-2 119,6 1,83 1,69
PR4-3 135,4 1,84 1,83
PR4-4 99,2 1,86 1,85
PR4-5 90,9 1,84 1,67
Lys+RBS-1 33,1 1,99 1,87
Lys+RBS-2 43,8 1,94 1,91
Lys+RBS-3 27,7 1,98 1,82
Lys+RBS-4 38,9 2,02 1,92
Lys+RBS-5 31,0 2,03 1,92
Lys+RBS-6 39,9 2,00 1,84
PGEX6P1-a 65,9 1,94 2,02
PGEX6P1-b 65,3 1,90 1,98
PGEX6P1-c 70,6 1,95 2,05
PGEX6P1-d 43,6 1,82 1,40
PGEX6P1-e 53,9 1,87 1,75
S54 219,6 1,87 2,21
S54 216,4 1,88 2,21


Precipitator

Cloning

Investigators: Sophie
continue from experiment: Miniprep (Jakob/Ruediger, 12.8., see lysis cassette)
project name: inducible promoter for Pbd
parts: S54 (Eco & Spe)
GFP-Pbd ε5 (Xba 7 Pst)
psb1t3 (Eco & Pst)
stored in: Ruediger's box

Ligation

Investigators: Sophie
continue from experiment: Cloning (today, me)
project name: inducible promoter for Pbd
parts: S54 (Eco & Spe)
GFP-Pbd ε5 (Xba 7 Pst)
psb1t3 (Eco & Pst)
name of ligation product: IPTG+Pbd
stored in: Ruediger's box

Testdigest

Investigators: Sophie
continue from experiment: Ligation (today, me)
project name: inducible promoter for Pbd
Freiburg 12.8.11.jpg PR6 6 and Pr 4 4 were sequenced but PRs were missing again. Inducible promoter seems to be necessary.

                       

Retrieved from "http://2011.igem.org/Team:Freiburg/Notebook/12_August"