Team:Groningen/Notebook/24 August 2009

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(Difference between revisions)
(New page: {{Team:Groningen/Notebook/Day/Header}} ==Wet== ===GVP Cluster=== :→ {{todo}} Clone GVP cluster behind Lac and Arabinose promoter for expression tests in pSB1AC3 plasmid :→ {{todo}} ...)
(Metal Accumulation)
 
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===GVP Cluster===
===GVP Cluster===
 +
:→ {{todo}} Restriction of GVP and pSB1AC3-Lac/pBAD for assembly
 +
:→ {{todo}} Gel purification of wanted fragments and nanodrop
:→ {{todo}} Clone GVP cluster behind Lac and Arabinose promoter for expression tests in pSB1AC3 plasmid
:→ {{todo}} Clone GVP cluster behind Lac and Arabinose promoter for expression tests in pSB1AC3 plasmid
:→ {{todo}} Send plasmids with ME<sup>tal</sup> promoters in BBa_J61002 vector for sequencing
:→ {{todo}} Send plasmids with ME<sup>tal</sup> promoters in BBa_J61002 vector for sequencing
Line 11: Line 13:
:→ {{todo}} Test promoter strenght compared to BBa_J23101 promoter (Sven)
:→ {{todo}} Test promoter strenght compared to BBa_J23101 promoter (Sven)
:→ {{todo}} Enter sequences of constructs to Sandbox
:→ {{todo}} Enter sequences of constructs to Sandbox
 +
 +
 +
[[Image:Plasmid Miniprep Kit Flow Protocol.gif|thumb|150px| www.sigmaaldrich.com]]
 +
 +
'''Plasmid Purification'''
 +
 +
Plasmid isolation was performed on the cultures of E.coli TOP10 containing plasmids [http://partsregistry.org/wiki/index.php?title=Part:BBa_J61002 BBa_J61002] with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K190016 Zinc+RBS], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K190017 Copper+RBS] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K190015 Arsenic+RBS] promoters and RFP with the "Sygma-Aldrich™ [http://www.sigmaaldrich.com/life-science/molecular-biology/dna-and-rna-purification/plasmid-miniprep-kit.html GenElute™] Plasmid Miniprep Kit".
 +
 +
* From each tube 4mL of culture was collected in a 2.0mL cup, and the cells were pelleted by centrifugation for 1 min. at max. speed and the supernatant discarded.
 +
* Plasmids were eluted with 30μL MQ and stored in the fridge
 +
 +
'''Restriction Control'''
 +
 +
Isolated plasmids were cut with fast-digest enzymes EcoRI and PstI to cut out promoter-RFP, and create ~2000bp and ~950bp fragments.
 +
 +
[[Image:24-8 no.1.jpg|400px]] [[Image:Generulers_1kb_marker_Fermentas.jpg]]
 +
 +
:→ From left to right: 1kB ladder, pArsR (4x), pCueO (3x), pZntR (3x)
 +
 +
:→ The concentration of plasmids was not determined before restriction was done, and likely the concentration was to high to give a band at ~3000bp of uncut plasmid.
 +
 +
 +
[[Image:24-8 no.2.jpg|700px]]
 +
 +
:→ From left to right: 1kB ladder, pArsR (2x), pCueO (2x), pZntR (2x), Control BBa_J23101, Empty Slot
 +
 +
:→ The 2% Agarose gel was run for 15 min. at 100V to get separation at low bp fragments, larger parts are grouped together at the top
 +
 +
:→ The expected sizes are seen in the gel, plasmids can be send for sequencing!
===Transporters===
===Transporters===
===Metal Accumulation===
===Metal Accumulation===
 +
*'''Check pSB1AC3-mymT, fmt, smtA'''
 +
**Pick 3 colonies of ''E. coli'' TOP10 + pSB1AC3-mymT, fmt, smtA.
 +
**Add 1uL MQ and microwave 1min to lyse the cells.
 +
**Do colony PCR with VR/VF primers and RBS-fw/gene specific rev primers.
 +
**Run PCR products on gel.
 +
[[Image:F102471 2009-08-24 Colony PCR fMT SmtA MymT.JPG|350px]]
 +
**From left to right:
 +
::Upper: SmtA-1 till 3 (1200bp), fMT 4-6 (500bp), MymT 7-8(500), AC3 (315bp) (using VF2/VR primers)
 +
::Lower: SmtA-1 till 3 (900bp), fMT 4-6 (200bp), MymT 7-9(215bp) (using gene specific primers)
 +
**Thereby the construct of Mymt-pSB1AC3 nr7-9 do not give correct fragment size, also the constructs pSB1AC3-SmtA nr 1 and pSB1AC3-fMT nr 5 do not give correct fragment size.
 +
**Put o/n culture @ warm room of pSB1AC3-SmtA nr2&3 and pSB1AC3-fMT nr 4&6.
 +
 +
*'''Cloning of MymT into pSB1AC3 or other vectors has stopped'''
 +
**It is considered to be to much time consuming to also clone MymT next to the other MTs.
===Vectors===
===Vectors===
 +
====Metal promotors====
 +
[http://wolfson.huji.ac.il/expression/vector/origin.html#top origin of replication] determines:
 +
* Vector copy number
 +
* Plasmid compatibility: its ability to replicate in conjuction with another plasmid.
 +
** Plasmids that utilize the same replication system cannot co-exist in the same bacterial cell.
 +
** pMB1<sup>*</sup> and ColE1 are in different [http://wolfson.huji.ac.il/expression/vector/origin.html#comp compatibility group]
 +
** pMB1 and ColE1 are too similar and thus not compatible
 +
<sup>*</sup> pMB1 is the pUC19 derived ori present in pSB1AC3
==Dry==
==Dry==
{{Team:Groningen/Notebook/Day/Footer}}
{{Team:Groningen/Notebook/Day/Footer}}

Latest revision as of 16:48, 25 August 2009

Igemhomelogo.png

Wet

GVP Cluster

TODO Restriction of GVP and pSB1AC3-Lac/pBAD for assembly
TODO Gel purification of wanted fragments and nanodrop
TODO Clone GVP cluster behind Lac and Arabinose promoter for expression tests in pSB1AC3 plasmid
TODO Send plasmids with MEtal promoters in BBa_J61002 vector for sequencing
TODO Check MEtal + RBS promoters on gel with restriction (also XbaI/PstI in Zinc promoter)
TODO Test promoter strenght compared to BBa_J23101 promoter (Sven)
TODO Enter sequences of constructs to Sandbox


www.sigmaaldrich.com

Plasmid Purification

Plasmid isolation was performed on the cultures of E.coli TOP10 containing plasmids BBa_J61002 with Zinc+RBS, Copper+RBS and Arsenic+RBS promoters and RFP with the "Sygma-Aldrich™ GenElute™ Plasmid Miniprep Kit".

  • From each tube 4mL of culture was collected in a 2.0mL cup, and the cells were pelleted by centrifugation for 1 min. at max. speed and the supernatant discarded.
  • Plasmids were eluted with 30μL MQ and stored in the fridge

Restriction Control

Isolated plasmids were cut with fast-digest enzymes EcoRI and PstI to cut out promoter-RFP, and create ~2000bp and ~950bp fragments.

24-8 no.1.jpg Generulers 1kb marker Fermentas.jpg

→ From left to right: 1kB ladder, pArsR (4x), pCueO (3x), pZntR (3x)
→ The concentration of plasmids was not determined before restriction was done, and likely the concentration was to high to give a band at ~3000bp of uncut plasmid.


24-8 no.2.jpg

→ From left to right: 1kB ladder, pArsR (2x), pCueO (2x), pZntR (2x), Control BBa_J23101, Empty Slot
→ The 2% Agarose gel was run for 15 min. at 100V to get separation at low bp fragments, larger parts are grouped together at the top
→ The expected sizes are seen in the gel, plasmids can be send for sequencing!

Transporters

Metal Accumulation

  • Check pSB1AC3-mymT, fmt, smtA
    • Pick 3 colonies of E. coli TOP10 + pSB1AC3-mymT, fmt, smtA.
    • Add 1uL MQ and microwave 1min to lyse the cells.
    • Do colony PCR with VR/VF primers and RBS-fw/gene specific rev primers.
    • Run PCR products on gel.

F102471 2009-08-24 Colony PCR fMT SmtA MymT.JPG

    • From left to right:
Upper: SmtA-1 till 3 (1200bp), fMT 4-6 (500bp), MymT 7-8(500), AC3 (315bp) (using VF2/VR primers)
Lower: SmtA-1 till 3 (900bp), fMT 4-6 (200bp), MymT 7-9(215bp) (using gene specific primers)
    • Thereby the construct of Mymt-pSB1AC3 nr7-9 do not give correct fragment size, also the constructs pSB1AC3-SmtA nr 1 and pSB1AC3-fMT nr 5 do not give correct fragment size.
    • Put o/n culture @ warm room of pSB1AC3-SmtA nr2&3 and pSB1AC3-fMT nr 4&6.
  • Cloning of MymT into pSB1AC3 or other vectors has stopped
    • It is considered to be to much time consuming to also clone MymT next to the other MTs.

Vectors

Metal promotors

origin of replication determines:

  • Vector copy number
  • Plasmid compatibility: its ability to replicate in conjuction with another plasmid.
    • Plasmids that utilize the same replication system cannot co-exist in the same bacterial cell.
    • pMB1* and ColE1 are in different compatibility group
    • pMB1 and ColE1 are too similar and thus not compatible

* pMB1 is the pUC19 derived ori present in pSB1AC3

Dry

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