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Team:Groningen/Notebook/10 September 2009
From 2009.igem.org
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(New page: {{Team:Groningen/Notebook/Day/Header}} ==Wet== ===GVP Cluster=== '''O.n. precultures''' :→ All tubes for plasmid isolation (pLacI (pSB1A2)(no.1 and no.2), pSB2K3 (12J)(2x) and pSB2K3...) |
(→GVP Cluster) |
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:→ All tubes for plasmid isolation (pLacI (pSB1A2)(no.1 and no.2), pSB2K3 (12J)(2x) and pSB2K3 (12L)(2x) showed growth. The pSB2K3 had less growth (and smaller pellet) than the other tubes. | :→ All tubes for plasmid isolation (pLacI (pSB1A2)(no.1 and no.2), pSB2K3 (12J)(2x) and pSB2K3 (12L)(2x) showed growth. The pSB2K3 had less growth (and smaller pellet) than the other tubes. | ||
| + | |||
| + | [[Image:Plasmid Miniprep Kit Flow Protocol.gif|thumb|150px| www.sigmaaldrich.com]] | ||
'''Plasmid isolation''' | '''Plasmid isolation''' | ||
| + | Plasmid isolation was performed on the cultures of E.coli TOP10 containing the above mentioned plasmids 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 (tubes from pSB2K3 12J and 12L were combined), 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 | ||
| + | |||
| + | |||
| + | '''Concentrations''' | ||
| + | |||
| + | {|cellpadding="2" cellspacing="1" border="4" | ||
| + | |'''Plasmid''' | ||
| + | |'''Conc. ng/μL''' | ||
| + | |'''260/280 | ||
| + | |'''260/230 ''' | ||
| + | |'''-20 box (michael | ||
| + | |'''Restriction Control''' | ||
| + | |- | ||
| + | |pLacI no.1 (pSB1A2) | ||
| + | |66.3 | ||
| + | |1.86 | ||
| + | |1.82 | ||
| + | |x | ||
| + | |Yes (SpeI/PstI) | ||
| + | |- | ||
| + | |pLacI no.2 (pSB1A2) | ||
| + | |47.1 | ||
| + | |1.82 | ||
| + | |1.45 | ||
| + | |x | ||
| + | |Yes (SpeI/PstI) | ||
| + | |- | ||
| + | |pSB2K3 (12J) | ||
| + | |142.9 | ||
| + | |1.89 | ||
| + | |2.31 | ||
| + | |x | ||
| + | |Yes (EcoRI/PstI) | ||
| + | |- | ||
| + | |pSB2K3 (12L) | ||
| + | |337.1 | ||
| + | |1.87 | ||
| + | |2.35 | ||
| + | |x | ||
| + | |Yes (EcoRI/PstI) | ||
| + | |} | ||
| Line 86: | Line 130: | ||
| - | [[Image:10-9 no.1.jpg| | + | [[Image:10-9 no.1.jpg|340px]] [[Image:Generulers_1kb_marker_Fermentas.jpg]] |
| + | |||
| + | :→ From left to right: 1kB ladder, pSB2K3 12J (2x), Empty Slot, pSB2K3 12L (2x), Empty Slot, pZntR-GVP (2x) | ||
| + | |||
| + | [[Image:10-9 no.2.jpg|280px]] [[Image:Generulers_1kb_marker_Fermentas.jpg]] | ||
| + | |||
| + | :→ From left to right: 1kB ladder, pLacI no.1 (pSB1A2)(2x), pLacI no.2 (pSB1A2)(2x), GVP (2x) | ||
| + | |||
| + | |||
| + | '''Purification''' | ||
| + | |||
| + | [[Image:Zymoclean Gel DNA Recovery Kit (D4001) 2.jpg|thumb|150px| www.zymoresearch.com]] | ||
| + | |||
| + | |||
| + | |||
| + | :→ In step 7 the fragments were eluted in 12μL MQ and was stored on ice until use. | ||
| + | |||
| + | |||
| + | '''Concentration''' | ||
| + | |||
| + | |||
| + | '''Ligation''' | ||
| + | |||
| + | A total amount of vector of 100ng was used in a 1:3 ratio with insert. | ||
| + | |||
| + | :* 3 uL Ligase buffer | ||
| + | :* 1 ul T4 Ligase | ||
| + | :* uL plasmid pSB1A2 pLacI digested with SpeI and PstI | ||
| + | :* uL GVP restricted with XbaI and PstI | ||
| + | |||
| + | |||
| + | :* 3 uL Ligase buffer | ||
| + | :* 1 ul T4 Ligase | ||
| + | :* uL plasmid pSB2K3 digested with EcoRI and PstI | ||
| + | :* uL pZntR-GVP and pCueO-GVP restricted with EcoRI and PstI | ||
| + | :* uL MQ | ||
| + | |||
| + | ''Incubate:'' | ||
| + | :* 25°C 50min. | ||
| + | :* kept on ice for 10min. | ||
| + | |||
| + | '''Tranformation''' | ||
| + | :* add 10uL of the ligation product to 50uL competent E.coli TOP10 cells. | ||
| + | ''Incubate:'' | ||
| + | :* 30 min @ ice | ||
| + | :* 90 sec 42°C | ||
| + | :* 2 min @ ice | ||
| + | :* add 800uL LB-medium | ||
| + | :* incubate for 1 h at 37°C | ||
| + | :* plate on LB-amp<sub>100</sub> plates | ||
| + | |||
| - | :→ | + | :→ Positive control was BBa_J61002-J23101 plasmid, and negative control was MQ. |
| - | |||
| - | + | '''Observations''' | |
| + | Here are the allignments of the expect | ||
===Transporters=== | ===Transporters=== | ||
===Metal Accumulation=== | ===Metal Accumulation=== | ||
| + | ====Alignment fMT==== | ||
| + | <center>[[Image:Allignments_fMT.jpg]]</center> | ||
| + | Alignments of the metallothionein fMT constructs, above is <i>In silico</i> construct, below is sequence determined with VF2 primer. The first is without promotor (<partinfo>BBa_K190019</partinfo>), second with constitutive promotor <partinfo>BBa_J23109</partinfo> (<partinfo>BBa_K190031</partinfo>) and the third with IPTG inducible promotor <partinfo>BBa_R0010</partinfo> (<partinfo>BBa_K190032</partinfo>). RBS is <partinfo>BBa_B0034</partinfo>. In combining the promotor and the coding sequence the Partregistry seems to add two unexpected additional bases (ag) between the two, missing in sequenced sample. In preparing the <i>In silico</i> construct a different prefix (lacking two bases (ag)), seen in sequenced samples, was used. The n's in the suffix of <partinfo>BBa_K190032</partinfo> were determined to be a g as expected when giving a closer look into the chromatogram. | ||
| + | ====Alignment MBP-ArsR==== | ||
| + | <center>[[Image:Allignment_MBP-ArsR.jpg|750px]]</center> | ||
| + | Alignments of the metal chelator Maltose Binding Protein (MBP)-ArsR fusion protein (<partinfo>BBa_K190027</partinfo>). Above is <i>In silico</i> construct, in the middle is sequence determined with VF2 primer, below the sequence determined with VR primer. A Tobacco Etch Virus (TEV) protease cleavage site is added between the two proteins. | ||
===Vectors=== | ===Vectors=== | ||
Latest revision as of 12:37, 14 September 2009
[http://2009.igem.org/Team:Groningen http://2009.igem.org/wiki/images/f/f1/Igemhomelogo.png]
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Wet
GVP Cluster
O.n. precultures
- → All tubes for plasmid isolation (pLacI (pSB1A2)(no.1 and no.2), pSB2K3 (12J)(2x) and pSB2K3 (12L)(2x) showed growth. The pSB2K3 had less growth (and smaller pellet) than the other tubes.
Plasmid isolation
Plasmid isolation was performed on the cultures of E.coli TOP10 containing the above mentioned plasmids 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 (tubes from pSB2K3 12J and 12L were combined), 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
Concentrations
| Plasmid | Conc. ng/μL | 260/280 | 260/230 | -20 box (michael | Restriction Control |
| pLacI no.1 (pSB1A2) | 66.3 | 1.86 | 1.82 | x | Yes (SpeI/PstI) |
| pLacI no.2 (pSB1A2) | 47.1 | 1.82 | 1.45 | x | Yes (SpeI/PstI) |
| pSB2K3 (12J) | 142.9 | 1.89 | 2.31 | x | Yes (EcoRI/PstI) |
| pSB2K3 (12L) | 337.1 | 1.87 | 2.35 | x | Yes (EcoRI/PstI) |
Restriction for Assembly
The plasmids from the o.n. precultures were cut with PstI and EcoRI to cut out the entire part between the pre- and suffix.
| Plasmid | Amount μL | MQ μL | Fast digest buffer | EcoRI fast digest enzyme | XbaI fast digest enzyme | SpeI fast digest enzyme | PstI fast digest enzyme |
| pLacI no.1 (pSB1A2) | 16.0 | x | 3.0 | x | x | 1.0 | 1.0 |
| pLacI no.1 (pSB1A2) | 16.0 | x | 3.0 | x | x | 1.0 | 1.0 |
| GVP | 4.0 | 11.0 | 3.0 | x | 1.0 | x | 1.0 |
| pSB2K3 (12J) | 8.0 | 6.0 | 3.0 | 1.0 | x | x | 1.0 |
| pSB2K3 (12L) | 4.0 | 11.0 | 3.0 | 1.0 | x | x | 1.0 |
| pZntR-GVP | 12.0 | 3.0 | 3.0 | 1.0 | x | x | 1.0 |
- → From left to right: 1kB ladder, pSB2K3 12J (2x), Empty Slot, pSB2K3 12L (2x), Empty Slot, pZntR-GVP (2x)
- → From left to right: 1kB ladder, pLacI no.1 (pSB1A2)(2x), pLacI no.2 (pSB1A2)(2x), GVP (2x)
Purification
_2.jpg)
- → In step 7 the fragments were eluted in 12μL MQ and was stored on ice until use.
Concentration
Ligation
A total amount of vector of 100ng was used in a 1:3 ratio with insert.
- 3 uL Ligase buffer
- 1 ul T4 Ligase
- uL plasmid pSB1A2 pLacI digested with SpeI and PstI
- uL GVP restricted with XbaI and PstI
- 3 uL Ligase buffer
- 1 ul T4 Ligase
- uL plasmid pSB2K3 digested with EcoRI and PstI
- uL pZntR-GVP and pCueO-GVP restricted with EcoRI and PstI
- uL MQ
Incubate:
- 25°C 50min.
- kept on ice for 10min.
Tranformation
- add 10uL of the ligation product to 50uL competent E.coli TOP10 cells.
Incubate:
- 30 min @ ice
- 90 sec 42°C
- 2 min @ ice
- add 800uL LB-medium
- incubate for 1 h at 37°C
- plate on LB-amp100 plates
- → Positive control was BBa_J61002-J23101 plasmid, and negative control was MQ.
Observations
Here are the allignments of the expect
Transporters
Metal Accumulation
Alignment fMT
Alignments of the metallothionein fMT constructs, above is In silico construct, below is sequence determined with VF2 primer. The first is without promotor (), second with constitutive promotor () and the third with IPTG inducible promotor (). RBS is . In combining the promotor and the coding sequence the Partregistry seems to add two unexpected additional bases (ag) between the two, missing in sequenced sample. In preparing the In silico construct a different prefix (lacking two bases (ag)), seen in sequenced samples, was used. The n's in the suffix of were determined to be a g as expected when giving a closer look into the chromatogram.
Alignment MBP-ArsR
Alignments of the metal chelator Maltose Binding Protein (MBP)-ArsR fusion protein (). Above is In silico construct, in the middle is sequence determined with VF2 primer, below the sequence determined with VR primer. A Tobacco Etch Virus (TEV) protease cleavage site is added between the two proteins.
Vectors
Dry
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