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Team:Groningen/Notebook/31 July 2009
From 2009.igem.org
(Difference between revisions)
(New page: {{Team:Groningen/Notebook/Day/Header}} ==Wet== ===GVP Cluster=== ===Transporters=== ===Metal Accumulation=== '''ArsR-MBP fusion''' ::colony PCR MBP ::: ::Overnight culture MBP ===V...) |
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===Transporters=== | ===Transporters=== | ||
| + | '''GlpF''' | ||
| + | *Restriction/ligase and transformation of GlpF in psB1AC3 | ||
| + | |||
| + | : '''Restriction''' | ||
| + | {| | ||
| + | |10ul | ||
| + | |GlpF PCR | ||
| + | |- | ||
| + | |2 ul | ||
| + | |Buffer | ||
| + | |- | ||
| + | |05.ul | ||
| + | |EcoRI | ||
| + | |- | ||
| + | |0.5 ul | ||
| + | |SpeI | ||
| + | |- | ||
| + | |7 uL | ||
| + | |MQ | ||
| + | |- | ||
| + | |} | ||
| + | |||
| + | :*37 30min | ||
| + | :* Inactivation on Gel | ||
| + | :* Extraction from gel | ||
| + | |||
| + | |||
| + | :'''Ligation''' | ||
| + | {| | ||
| + | |2 uL | ||
| + | |Ligase buffer | ||
| + | | | ||
| + | |rowspan="4" style="border-left:solid 1px"|16° 1h → 65° 10 min | ||
| + | |- | ||
| + | |1 ul | ||
| + | |T4 Ligase | ||
| + | | | ||
| + | |- | ||
| + | |11 uL | ||
| + | |GlpF digested with EcoRI and SpeI | ||
| + | |rowspan="2" style="border-left:solid 1px"|42° 2 min → | ||
| + | |- | ||
| + | |11 uL | ||
| + | |psB1AC3 digested with EcoRi and SpeI | ||
| + | |- | ||
| + | |} | ||
| + | |||
| + | :'''Tranformation''' | ||
| + | ::* add 4 uL of the GlpFpsB1AC3 ligation mixture to 50uL competent e.coli top10 cells. | ||
| + | Incubate: | ||
| + | :::*30 min @ ice | ||
| + | :::*5 min 37° | ||
| + | :::*5min @ ice | ||
| + | ::*add 800uL LB | ||
| + | :::* incubate for 1 h at 37° | ||
| + | ::* plate on LB-AMP plates | ||
===Metal Accumulation=== | ===Metal Accumulation=== | ||
'''ArsR-MBP fusion''' | '''ArsR-MBP fusion''' | ||
| - | ::colony PCR MBP | + | ::*'''colony PCR MBP:''' |
| - | + | {| | |
| - | + | | | |
| + | {| border="1" | ||
| + | |+ '''colony PCR MBP''' | ||
| + | ! Component !! amount | ||
| + | |- | ||
| + | ! 2x Phusion MM | ||
| + | | 12.5 uL | ||
| + | |- | ||
| + | ! MQ | ||
| + | | 9.5 uL | ||
| + | |- | ||
| + | ! MBP Fw | ||
| + | | 1 uL | ||
| + | |- | ||
| + | ! MBP Rev | ||
| + | | 1 uL | ||
| + | |- | ||
| + | ! Colony MBP | ||
| + | | 1 uL | ||
| + | |} | ||
| + | |width="15"| | ||
| + | | | ||
| + | <!--Tabel 2 hier--> | ||
| + | {| | ||
| + | ! Touchdown MBP program | ||
| + | ! Temperature | ||
| + | ! Time | ||
| + | |- | ||
| + | |Denaturing | ||
| + | |98° | ||
| + | |2.00 min | ||
| + | |- | ||
| + | | | ||
| + | |Touchdown 10X 60->50 | ||
| + | |- | ||
| + | |Denaturing | ||
| + | |98° | ||
| + | |30 sec | ||
| + | |- | ||
| + | |Annealing | ||
| + | |60°->50° | ||
| + | |30 sec | ||
| + | |- | ||
| + | |Elongation | ||
| + | |72° | ||
| + | |30 sec | ||
| + | |- | ||
| + | | | ||
| + | |End cycles | ||
| + | |-| | ||
| + | |Start Cycles 25X | ||
| + | |- | ||
| + | |Denaturing | ||
| + | |98° | ||
| + | |30sec | ||
| + | |- | ||
| + | |Annealing | ||
| + | |60° | ||
| + | |30 sec | ||
| + | |- | ||
| + | |Elongation | ||
| + | |72° | ||
| + | |30 sec | ||
| + | |- | ||
| + | | | ||
| + | |End cycles | ||
| + | |- | ||
| + | |Final elongation | ||
| + | |72° | ||
| + | |7 min | ||
| + | |- | ||
| + | |Hold | ||
| + | |4° | ||
| + | |Forever | ||
| + | |} | ||
| + | |width="10"| | ||
| + | |} | ||
| + | ::*'''Overnight culture MBP''' | ||
===Vectors=== | ===Vectors=== | ||
| + | *Metal sensitive promoters | ||
| + | Ligations were transformed into a [http://openwetware.org/wiki/User:Wilfred_J._Poppinga/Notebook/Wilfreds_Project/2009/07/31#Competent_cells new batch] of competent cells and plated out on TY Amp<sub>100</sub> plates. | ||
| + | |||
| + | *'''Colonies of pSB3K3-HML and pSB1AC3-HML + RFP, and pSB3K3 or pSB1AC3 + pLac''' | ||
| + | **pSB3K3-H, M, L + RFP all had colonies (not in all concentrations) | ||
| + | **pSB1AC3-H, M, L + RFP all had colonies | ||
| + | **pSB3K3 and pSB1AC3 + pLac had no colonies at all... | ||
| + | |||
| + | *'''PCR to check pSB3K3-HML and pSB1AC3-HML + RFP''' | ||
| + | **Pick 2 colonies per construct (put 1uL MQ in PCR tube and resuspend) | ||
| + | **Boil 1 min @ microwave (max power) | ||
| + | **Add MM and polymerase | ||
| + | **Run programm: Colony | ||
| + | **Expected size: 1420bp | ||
| + | **Run products on 1% Agarose gel (unfortunately the gel was bad so the size could not be estimated) | ||
| + | **Bands were seen for KH1&2, KM1&2, KL1, AH1&2, AL1. | ||
==Dry== | ==Dry== | ||
| + | We had a look at ways to measure things concerning gas vesicles (buoyant density, volume, etc.), buoyant density was [http://dx.doi.org/10.1016/0167-7012(92)90048-9 previously measured] using a specific apparatus which we're not likely to obtain. We also talked with some biologists about ways to detect protein concentrations (or rather, amounts) using antibodies, apparently this might be possible if we attach something for which there is a kind of "standard" antibody available (and the maltose binding protein we're attaching to ArsR ''might'' qualify as such). | ||
| + | |||
| + | To measure the total arsenic content in the cell you can use coupled mass spectrometry. for more details see Metal accumulation. | ||
{{Team:Groningen/Notebook/Day/Footer}} | {{Team:Groningen/Notebook/Day/Footer}} | ||
Latest revision as of 10:22, 5 August 2009
[http://2009.igem.org/Team:Groningen http://2009.igem.org/wiki/images/f/f1/Igemhomelogo.png]
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|---|
Wet
GVP Cluster
Transporters
GlpF
- Restriction/ligase and transformation of GlpF in psB1AC3
- Restriction
| 10ul | GlpF PCR |
| 2 ul | Buffer |
| 05.ul | EcoRI |
| 0.5 ul | SpeI |
| 7 uL | MQ |
- 37 30min
- Inactivation on Gel
- Extraction from gel
- Ligation
| 2 uL | Ligase buffer | 16° 1h → 65° 10 min | |
| 1 ul | T4 Ligase | ||
| 11 uL | GlpF digested with EcoRI and SpeI | 42° 2 min → | |
| 11 uL | psB1AC3 digested with EcoRi and SpeI |
- Tranformation
- add 4 uL of the GlpFpsB1AC3 ligation mixture to 50uL competent e.coli top10 cells.
Incubate:
- 30 min @ ice
- 5 min 37°
- 5min @ ice
- add 800uL LB
- incubate for 1 h at 37°
- plate on LB-AMP plates
Metal Accumulation
ArsR-MBP fusion
- colony PCR MBP:
|
|
- Overnight culture MBP
Vectors
- Metal sensitive promoters
Ligations were transformed into a [http://openwetware.org/wiki/User:Wilfred_J._Poppinga/Notebook/Wilfreds_Project/2009/07/31#Competent_cells new batch] of competent cells and plated out on TY Amp100 plates.
- Colonies of pSB3K3-HML and pSB1AC3-HML + RFP, and pSB3K3 or pSB1AC3 + pLac
- pSB3K3-H, M, L + RFP all had colonies (not in all concentrations)
- pSB1AC3-H, M, L + RFP all had colonies
- pSB3K3 and pSB1AC3 + pLac had no colonies at all...
- PCR to check pSB3K3-HML and pSB1AC3-HML + RFP
- Pick 2 colonies per construct (put 1uL MQ in PCR tube and resuspend)
- Boil 1 min @ microwave (max power)
- Add MM and polymerase
- Run programm: Colony
- Expected size: 1420bp
- Run products on 1% Agarose gel (unfortunately the gel was bad so the size could not be estimated)
- Bands were seen for KH1&2, KM1&2, KL1, AH1&2, AL1.
Dry
We had a look at ways to measure things concerning gas vesicles (buoyant density, volume, etc.), buoyant density was [http://dx.doi.org/10.1016/0167-7012(92)90048-9 previously measured] using a specific apparatus which we're not likely to obtain. We also talked with some biologists about ways to detect protein concentrations (or rather, amounts) using antibodies, apparently this might be possible if we attach something for which there is a kind of "standard" antibody available (and the maltose binding protein we're attaching to ArsR might qualify as such).
To measure the total arsenic content in the cell you can use coupled mass spectrometry. for more details see Metal accumulation.
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