Team:Groningen/Notebook/18 September 2009

From 2009.igem.org

(Difference between revisions)
(New page: {{Team:Groningen/Notebook/Day/Header}} ==Wet== ===GVP Cluster=== '''Plates''' The ampicillin stocks showed signs of failure (growth on negative control, overgrowth of low concentration...)
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===GVP Cluster===
===GVP Cluster===
-
'''Plates'''
+
'''Planning'''
-
The ampicillin stocks showed signs of failure (growth on negative control, overgrowth of low concentration plates, etc.), and therefor a new batch was made and used for agar plates to check if the ampicillin works. A positive and negative transformation with the new competent cells was performed and plated on LB-agar-amp<sub>100</sub> plates in low and high concentration.
+
:→ {{todo}} work out the wiki page for GVP
 +
::* made a layout
 +
::* still have to read all articles
 +
::* modeling will stay as it is, has been done by modeling people
 +
:→ {{done}} make a doodle for presentation planning (1-19 oct.)
 +
:→ {{done}} media attention
 +
::* mail to UK, Ing., St. Gen.
 +
::* facebook account with link to twitter
 +
::* ethics survey link on facebook and twitter
 +
:→ {{done}} place an ethics survey link on twitter
 +
 
 +
 
 +
:→ {{done}} clone pArsR-GVP into pSB2K3
 +
::* {{todo}} still need to make glycerol stock
 +
:→ {{todo}} clone repeat out of GVP cluster
 +
:→ {{todo}} make glycerol stocks of constructs
 +
:→ {{todo}} enter info on part registry
 +
 
 +
 
 +
'''Plates'''
{|cellpadding="2" cellspacing="1" border="4"
{|cellpadding="2" cellspacing="1" border="4"
Line 15: Line 34:
|'''No. of Colonies'''
|'''No. of Colonies'''
|'''Date'''
|'''Date'''
-
|-
 
-
|Positive (low)
 
-
|J61002 (with J23101)
 
-
|Ampicillin
 
-
|~200 (red)
 
-
|11 sept.
 
|-
|-
|Positive (high)
|Positive (high)
|J61002 (with J23101)
|J61002 (with J23101)
|Ampicillin
|Ampicillin
-
|~600 (red)
+
|~1500 (red)
-
|11 sept.
+
|17 sept.
|-
|-
-
|Negative (low)
+
|Negative (high)
|None
|None
|None
|None
 +
|8 (white/yellow)
 +
|17 sept.
 +
|-
 +
|pMA-gvpL (low)
 +
|pMA
 +
|Ampicillin
 +
|~150
 +
|17 sept.
 +
|-
 +
|pMA-gvpL (high)
 +
|pMA
 +
|Ampicillin
 +
|~650
 +
|17 sept.
 +
|-
 +
|pMA-GlpF (low)
 +
|pMA
 +
|Ampicillin
 +
|~125
 +
|17 sept.
 +
|-
 +
|pMA-gvpL (high)
 +
|pMA
 +
|Ampicillin
 +
|~550
 +
|17 sept.
 +
|-
 +
|pNL29 (SJ)(low)
 +
|pBlueScript_II_KS_(+)
 +
|Ampicillin
 +
|~100
 +
|17 sept.
 +
|-
 +
|pNL29 (high)
 +
|pBlueScript_II_KS_(+)
 +
|Ampicillin
 +
|~250
 +
|17 sept.
 +
|-
 +
|pArsR-GVP (on Ampicillin)
 +
|pSB2K3
 +
|Kanamycin
|0
|0
-
|11 sept.
+
|17 sept.
|-
|-
-
|Negative (high)
+
|pArsR-GVP (Glycerol Stock)
-
|None
+
|pSB2K3
-
|None
+
|Kanamycin
-
|3 (white/yellow)
+
|Single Colonies
-
|11 sept.
+
|17 sept.
 +
|-
 +
|pArsR-GVP (Saline Test)
 +
|pSB2K3
 +
|Kanamycin
 +
|Plate Full-Grown
 +
|17 sept.
 +
|-
 +
|pZntR-GVP (Saline Test)
 +
|pSB2K3
 +
|Kanamycin
 +
|Plate Full-Grown
 +
|17 sept.
 +
|-
 +
|pCueO-GVP (Saline Test)
 +
|pSB2K3
 +
|Kanamycin
 +
|Plate Full-Grown
 +
|17 sept.
|}
|}
-
:→ The plates showed the expected amount of colonies, and also red in the case of the positive plates. The three colonies on the negative plate can be taken into acount when plating transformed cells.
+
:→ The plates showed the expected amount of colonies, and also red in the case of the positive plates. The eight colonies on the negative plate can be taken into acount when plating transformed cells.
-
:→ The two plates with stripes of pZntR-GVP (pSB2K3) and pCueO-GVP (pSB2K3) colonies showed growth and some single colonies.
+
:→ The plate with stripes of pArsR-GVP (pSB2K3) preculture showed growth and some single colonies.
-
:→ All six plates were stored in the fridge for further use on monday.
+
:→ All plates were stored in the fridge for further use on monday.
'''Cultures'''
'''Cultures'''
-
The overnight cultures with LB-amp<sub>100</sub> medium of colonies E.coli TOP10 with pLacI-GVP in pSB1A2 (4x) and the positive and negative control plates, showed no growth.
+
The overnight cultures with LB-amp<sub>100</sub> medium of colonies E.coli TOP10 with pNL29 in pBlueScript_II_KS_(+) showed no growth.
-
 
+
-
 
+
-
:→ The lack of growth can be caused by the failing ampicillin stocks. The cells have probably lost their plasmid during growth on agar plates after transformation, due to the lack of need for ampicillin resistance. The colonies taken from the plates did not grow on the new ampicillin batch, and is a second conformation of our suspicions.
+
-
 
+
-
The overnight cultures with LB-kan<sub>50</sub> medium of colonies E.coli TOP10 with pZntR-GVP and pCueO-GVP in pSB2K3 (two of each) showed growth.
+
-
 
+
-
:→ From each culture 5mL was made into a pellet and stored in the -20 box of Michael. The pellets will be used for plasmid isolation on monday, and restriction to check if the GVP with promoter (~6000bp) and the vector (~4400bp) are as expected (self ligation would place the GVP construct in a ~3000bp vector.
 
 +
:→ The plate used for culture inoculation was very (more than a month) old, and was probably no good anymore. The new transformed E.coli TOP10 with original pNL29 plasmid will replace all excisting stocks/plates.
===Transporters===
===Transporters===

Latest revision as of 12:11, 18 September 2009

Igemhomelogo.png

Wet

GVP Cluster

Planning

TODO work out the wiki page for GVP
  • made a layout
  • still have to read all articles
  • modeling will stay as it is, has been done by modeling people
DONE make a doodle for presentation planning (1-19 oct.)
DONE media attention
  • mail to UK, Ing., St. Gen.
  • facebook account with link to twitter
  • ethics survey link on facebook and twitter
DONE place an ethics survey link on twitter


DONE clone pArsR-GVP into pSB2K3
  • TODO still need to make glycerol stock
TODO clone repeat out of GVP cluster
TODO make glycerol stocks of constructs
TODO enter info on part registry


Plates

Name Plasmid Used Antibiotics on Plasmid No. of Colonies Date
Positive (high) J61002 (with J23101) Ampicillin ~1500 (red) 17 sept.
Negative (high) None None 8 (white/yellow) 17 sept.
pMA-gvpL (low) pMA Ampicillin ~150 17 sept.
pMA-gvpL (high) pMA Ampicillin ~650 17 sept.
pMA-GlpF (low) pMA Ampicillin ~125 17 sept.
pMA-gvpL (high) pMA Ampicillin ~550 17 sept.
pNL29 (SJ)(low) pBlueScript_II_KS_(+) Ampicillin ~100 17 sept.
pNL29 (high) pBlueScript_II_KS_(+) Ampicillin ~250 17 sept.
pArsR-GVP (on Ampicillin) pSB2K3 Kanamycin 0 17 sept.
pArsR-GVP (Glycerol Stock) pSB2K3 Kanamycin Single Colonies 17 sept.
pArsR-GVP (Saline Test) pSB2K3 Kanamycin Plate Full-Grown 17 sept.
pZntR-GVP (Saline Test) pSB2K3 Kanamycin Plate Full-Grown 17 sept.
pCueO-GVP (Saline Test) pSB2K3 Kanamycin Plate Full-Grown 17 sept.


→ The plates showed the expected amount of colonies, and also red in the case of the positive plates. The eight colonies on the negative plate can be taken into acount when plating transformed cells.


→ The plate with stripes of pArsR-GVP (pSB2K3) preculture showed growth and some single colonies.


→ All plates were stored in the fridge for further use on monday.


Cultures

The overnight cultures with LB-amp100 medium of colonies E.coli TOP10 with pNL29 in pBlueScript_II_KS_(+) showed no growth.


→ The plate used for culture inoculation was very (more than a month) old, and was probably no good anymore. The new transformed E.coli TOP10 with original pNL29 plasmid will replace all excisting stocks/plates.

Transporters

Metal Accumulation

Vectors

Dry

April
MTWTFSS
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May
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June
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29 30
July
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27 28 29 30 31
August
MTWTFSS
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31
September
MTWTFSS
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21 22 23 24 25 26 27
28 29 30
October
MTWTFSS
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26 27 28 29 30 31
November
MTWTFSS
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30