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Team:Groningen/Notebook/26 August 2009
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m (New page: {{Team:Groningen/Notebook/Day/Header}} ==Wet== ===GVP Cluster=== ===Transporters=== ===Metal Accumulation=== <b>MBP-ArsR</b> *{{todo| Transform ligation with inducible promotors}} *{{t...) |
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===GVP Cluster=== | ===GVP Cluster=== | ||
| + | |||
| + | :→ {{done}} Restriction of GVP and pSB1AC3-Lac/pBAD for assembly | ||
| + | :→ {{done}} Gel purification of wanted fragments and nanodrop | ||
| + | :→ {{done}} Clone GVP cluster behind Lac and Arabinose promoter for expression tests in pSB1AC3 plasmid | ||
| + | |||
| + | :→ {{todo}} Test promoter strenght compared to BBa_J23101 promoter (Sven) | ||
| + | :→ {{todo}} Enter sequences of constructs to Sandbox | ||
| + | |||
| + | |||
| + | '''Restriction for Assembly''' | ||
| + | |||
| + | The vector [http://partsregistry.org/wiki/index.php?title=Part:pSB1AC3 pSB1AC3] containing the LAC and pBAD inducible promoters were cut with PstI and SpeI to create correct ends for insert of GVP biobrick [http://partsregistry.org/wiki/index.php?title=Part:BBa_I750016 BBa_I750016], which was cut with XbaI and PstI. | ||
| + | |||
| + | * 3 to 16μL plasmid in MQ (1.0μg) | ||
| + | * 13μL MQ (end volume of 20μL) | ||
| + | * 2μL Fast digest buffer | ||
| + | * 1μL PstI fast digest enzyme | ||
| + | * 1μL SpeI/XbaI fast digest enzyme | ||
| + | |||
| + | Restriction was kept at 37C for 30 min. and put on ice until used for gel purification. | ||
| + | |||
| + | '''Purification''' | ||
| + | |||
| + | [[Image:26-8 no.1.jpg|240px]] [[Image:Generulers_1kb_marker_Fermentas.jpg]] | ||
| + | |||
| + | :→ From left to right: 1kB ladder, pSB1AC3-LAC ,Empty Slot, pSB1AC3-pBAD-AraC , Empty Slot, GVP | ||
| + | |||
| + | :→ The band for the pSB1AC3-LAC vector was expected to be at ~3700bp (red line), but instead it was at ~2500bp. | ||
| + | |||
| + | :→ Comparison with the gel of Frans (constructed pSB1AC3-LAC) explained the band at a lower position (also seen on his gel), but not why it is so small. One option is use of pSB3K3 plasmid instead of pSB1AC3 plasmid, this would still give a band of ~3300bp and not ~2500bp! | ||
| + | |||
| + | |||
| + | A "Agarose Gel DNA Extraction Kit" [http://www.roche-applied-science.com/pack-insert/1696505a.pdf Standard Protocol] from Roche Applied Science. | ||
| + | |||
| + | :→ In step ten, 30μL MQ was added to the dry pellet and incubated at room temperature. | ||
| + | |||
| + | '''Concentrations''' | ||
| + | |||
| + | {|cellpadding="2" cellspacing="1" border="4" | ||
| + | |'''Plasmid''' | ||
| + | |'''Conc. ng/μL''' | ||
| + | |'''260/280 | ||
| + | |'''260/230 ''' | ||
| + | |'''-20 box (michael | ||
| + | |'''Restriction Control''' | ||
| + | |- | ||
| + | |pSB1AC3-LAC SpeI/PstI | ||
| + | |9.2 | ||
| + | |1.75 | ||
| + | |1.56 | ||
| + | |N.S. | ||
| + | |Gel | ||
| + | |- | ||
| + | |pSB1AC3-pBAD SpeI/PstI | ||
| + | |0.9 | ||
| + | |0.94 | ||
| + | |0.30 | ||
| + | |N.S. | ||
| + | |Gel | ||
| + | |- | ||
| + | |GVP XbaI/PstI restricted | ||
| + | |6.5 | ||
| + | |1.57 | ||
| + | |1.35 | ||
| + | |N.S. | ||
| + | |Gel | ||
| + | |} | ||
| + | |||
| + | '''Ligation''' | ||
| + | |||
| + | A total amount of vector of 50ng was used (GVP) in a 1:2 ratio with insert. | ||
| + | |||
| + | (1:2) (Done twice, old and new cut GVP insert) | ||
| + | :* 4 uL Ligase buffer | ||
| + | :* 2 ul T4 Ligase | ||
| + | :* 8 uL plasmid pSB1AC3-LAC/pBAD digested with PstI and SpeI | ||
| + | :* 8 uL insert GVP restricted with XbaI and PstI | ||
| + | |||
| + | (1:2) | ||
| + | :* 4 uL Ligase buffer | ||
| + | :* 2 ul T4 Ligase | ||
| + | :* 8 uL plasmid pSB3K3-L digested with PstI and SpeI | ||
| + | :* 8 uL insert GVP restricted with XbaI and PstI | ||
| + | |||
| + | ''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>/LB-kan<sub>50</sub> plates | ||
| + | |||
| + | |||
| + | :→ Positive control was BBa_J61002-J23101 plasmid, and negative control was MQ. | ||
===Transporters=== | ===Transporters=== | ||
Latest revision as of 13:07, 26 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
- → DONE Restriction of GVP and pSB1AC3-Lac/pBAD for assembly
- → DONE Gel purification of wanted fragments and nanodrop
- → DONE Clone GVP cluster behind Lac and Arabinose promoter for expression tests in pSB1AC3 plasmid
- → TODO Test promoter strenght compared to BBa_J23101 promoter (Sven)
- → TODO Enter sequences of constructs to Sandbox
Restriction for Assembly
The vector [http://partsregistry.org/wiki/index.php?title=Part:pSB1AC3 pSB1AC3] containing the LAC and pBAD inducible promoters were cut with PstI and SpeI to create correct ends for insert of GVP biobrick [http://partsregistry.org/wiki/index.php?title=Part:BBa_I750016 BBa_I750016], which was cut with XbaI and PstI.
- 3 to 16μL plasmid in MQ (1.0μg)
- 13μL MQ (end volume of 20μL)
- 2μL Fast digest buffer
- 1μL PstI fast digest enzyme
- 1μL SpeI/XbaI fast digest enzyme
Restriction was kept at 37C for 30 min. and put on ice until used for gel purification.
Purification
- → From left to right: 1kB ladder, pSB1AC3-LAC ,Empty Slot, pSB1AC3-pBAD-AraC , Empty Slot, GVP
- → The band for the pSB1AC3-LAC vector was expected to be at ~3700bp (red line), but instead it was at ~2500bp.
- → Comparison with the gel of Frans (constructed pSB1AC3-LAC) explained the band at a lower position (also seen on his gel), but not why it is so small. One option is use of pSB3K3 plasmid instead of pSB1AC3 plasmid, this would still give a band of ~3300bp and not ~2500bp!
A "Agarose Gel DNA Extraction Kit" [http://www.roche-applied-science.com/pack-insert/1696505a.pdf Standard Protocol] from Roche Applied Science.
- → In step ten, 30μL MQ was added to the dry pellet and incubated at room temperature.
Concentrations
| Plasmid | Conc. ng/μL | 260/280 | 260/230 | -20 box (michael | Restriction Control |
| pSB1AC3-LAC SpeI/PstI | 9.2 | 1.75 | 1.56 | N.S. | Gel |
| pSB1AC3-pBAD SpeI/PstI | 0.9 | 0.94 | 0.30 | N.S. | Gel |
| GVP XbaI/PstI restricted | 6.5 | 1.57 | 1.35 | N.S. | Gel |
Ligation
A total amount of vector of 50ng was used (GVP) in a 1:2 ratio with insert.
(1:2) (Done twice, old and new cut GVP insert)
- 4 uL Ligase buffer
- 2 ul T4 Ligase
- 8 uL plasmid pSB1AC3-LAC/pBAD digested with PstI and SpeI
- 8 uL insert GVP restricted with XbaI and PstI
(1:2)
- 4 uL Ligase buffer
- 2 ul T4 Ligase
- 8 uL plasmid pSB3K3-L digested with PstI and SpeI
- 8 uL insert GVP restricted with XbaI and PstI
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/LB-kan50 plates
- → Positive control was BBa_J61002-J23101 plasmid, and negative control was MQ.
Transporters
Metal Accumulation
MBP-ArsR
- Transform ligation with inducible promotors
- Prepare glycerol stock without promotors
Vectors
Dry
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