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Team:Groningen/Notebook/6 August 2009
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===Ideas to improve ligation=== | ===Ideas to improve ligation=== | ||
#Blunt end ligation of vector with PCR fragment, use Klenow polymerase to blunt end the vector. | #Blunt end ligation of vector with PCR fragment, use Klenow polymerase to blunt end the vector. | ||
| - | # | + | #Dephosphorylation of the vector via CAP. |
#Do not (preferably) clean up PCR product from gel, but use ~1uL for checking on gel and clean the rest with a PCR cleanup kit. | #Do not (preferably) clean up PCR product from gel, but use ~1uL for checking on gel and clean the rest with a PCR cleanup kit. | ||
#Use conventional restriction enzymes for digestion of the insert and vector, as these can be heat-inactivated. | #Use conventional restriction enzymes for digestion of the insert and vector, as these can be heat-inactivated. | ||
| Line 37: | Line 37: | ||
* 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. | * 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 20μL MQ and stored in the fridge | * Plasmids were eluted with 20μL MQ and stored in the fridge | ||
| + | |||
| + | '''Results: metal sensitive promoters in front of GVP''' | ||
| + | |||
| + | If the construct is correct one band at ~8kb should be visible. If the construct is incorrect one band at ~8kb and another at ~1.5kb should be visible. All plasmids have been cut with EcoRI and XbaI. | ||
| + | |||
| + | [[image:F102471_2009-08-06_15hr_36min_metal_sensitive_prom_+_GVP_(2).JPG]] | ||
| + | |||
| + | {| border="1" | ||
| + | |+'''''' | ||
| + | |1) 1kb ladder | ||
| + | |10) 1kb ladder | ||
| + | |- | ||
| + | |2) CueO- (1* concentrated) | ||
| + | |11) ZntR- (1* concentrated, diluted promoter) | ||
| + | |- | ||
| + | |3) ZntR- (1* concentrated) | ||
| + | |12) ArsR- (1* concentrated, diluted promoter) | ||
| + | |- | ||
| + | |4) ArsR- (1* concentrated) | ||
| + | |13) CueO- (9* concentrated, diluted promoter) | ||
| + | |- | ||
| + | |5) CueO- (9* concentrated) | ||
| + | |14) ZntR- (9* concentrated, diluted promoter) | ||
| + | |- | ||
| + | |6) ZntR- (9* concentrated) | ||
| + | |15) ArsR- (9* concentrated, diluted promoter) | ||
| + | |- | ||
| + | |7) ArsR- (9* concentrated) | ||
| + | |16 GVP vector (cut with EcoRI and XbaI) | ||
| + | |- | ||
| + | |8) CueO- (1* concentrated, diluted promoter) | ||
| + | |17) GVP vector (not digested) | ||
| + | |- | ||
| + | |9) 1kb ladder | ||
| + | |18) 1kb ladder | ||
| + | |} | ||
| + | |||
| + | The 1 and 9 times concentration refers to the amount of cells used to plate. Diluted promoter refers to the amount of promoter DNA used in the ligation (a hundred times less used than undiluted). | ||
| + | |||
===Transporters=== | ===Transporters=== | ||
| + | '''GlpF''' | ||
| + | |||
| + | Checking the previous ligation mixes by restriction with HaeII. HaeII cuts once in the psB1AC3 vector and once in the GlpF insert, so correct ligation results in two bands (2880 and 1019) if not a single band of 3055 is expected. HaeII cuts also twice in the ArsRMBP fusion (once in ArsR, once in MBP) and once in the psB1Ac3 vector so if the ArsRMBP insert is ligated correctly into the psB1Ac3 vector three bands are expected (507, 1068,2996) if not again a single band of 3055 is expected. | ||
| + | |||
| + | {| border="1" | ||
| + | |1 | ||
| + | |30/07/09 | ||
| + | |15uL | ||
| + | |GlpF cut with XbaI,PstI | ||
| + | |AC3 cut with EcoRI,SpeI | ||
| + | |- | ||
| + | |2 | ||
| + | |31/07/09 | ||
| + | |20uL | ||
| + | |GlpF cut with EcoRI,SpeI | ||
| + | |AC3 cut with EcoRI,SpeI | ||
| + | |- | ||
| + | |3 | ||
| + | |05/08/09 | ||
| + | |15uL | ||
| + | |GlpF cut with XbaI,PstI | ||
| + | |AC3 cut with XbaI,PstI | ||
| + | |- | ||
| + | |4 | ||
| + | |05/08/09 | ||
| + | |15uL | ||
| + | |ArsRMBP fusion cut with XbaI,PstI | ||
| + | |AC3 cut with XbaI,PstI | ||
| + | |- | ||
| + | |} | ||
| + | |||
| + | |||
| + | {| | ||
| + | |'''Restriction mix''' | ||
| + | | | ||
| + | | | ||
| + | |- | ||
| + | |8uL | ||
| + | |ligation | ||
| + | |rowspan="3" style="border-left:solid 1.5px"| → 37° 1.5h → 80° 20 min | ||
| + | |- | ||
| + | |1uL | ||
| + | |Tango | ||
| + | |- | ||
| + | |1uL | ||
| + | |HaeII | ||
| + | |} | ||
| + | |||
| + | '''PCR GlpF''' | ||
| + | |||
| + | Redone PCR 1 and PCR 2 | ||
| + | |||
| + | {| | ||
| + | | | ||
| + | {| | ||
| + | |''PCR1'' | ||
| + | |- | ||
| + | |25 uL | ||
| + | |Phusion mix | ||
| + | |- | ||
| + | |1 uL | ||
| + | |Forward GlpF | ||
| + | |- | ||
| + | |1 uL | ||
| + | | mutrev GlpF | ||
| + | |- | ||
| + | |0.5 ul | ||
| + | | K-12 genomic DNA | ||
| + | |- | ||
| + | |10 uL | ||
| + | |MQ | ||
| + | |- | ||
| + | |} | ||
| + | | | ||
| + | {| | ||
| + | |''PCR2'' | ||
| + | |- | ||
| + | |25 uL | ||
| + | |Phusion mix | ||
| + | |- | ||
| + | |1 uL | ||
| + | |Reverse GlpF | ||
| + | |- | ||
| + | |1 uL | ||
| + | | mutfwGlpF | ||
| + | |- | ||
| + | |0.5 ul | ||
| + | | K-12 genomic DNA | ||
| + | |- | ||
| + | |10 uL | ||
| + | |MQ | ||
| + | |- | ||
| + | |} | ||
| + | | | ||
| + | {| | ||
| + | |+''GlpF PCR1 and2 program'' | ||
| + | |Stage | ||
| + | |Temperature | ||
| + | |Time | ||
| + | |- | ||
| + | |Denaturing | ||
| + | |98° | ||
| + | |5 min | ||
| + | |- | ||
| + | |Touchdown | ||
| + | |60°-50° | ||
| + | |10 cycles | ||
| + | |- | ||
| + | | | ||
| + | |Start Cycles 30X | ||
| + | |- | ||
| + | |Denaturing | ||
| + | |98° | ||
| + | |30 sec | ||
| + | |- | ||
| + | |Annealing | ||
| + | |55° | ||
| + | |30 sec | ||
| + | |- | ||
| + | |Elongation | ||
| + | |72° | ||
| + | |30 sec | ||
| + | |- | ||
| + | | | ||
| + | |End cycles | ||
| + | |- | ||
| + | |Final elongation | ||
| + | |72° | ||
| + | |10 min | ||
| + | |- | ||
| + | |Hold | ||
| + | |4° | ||
| + | |Forever | ||
| + | |} | ||
| + | | | ||
| + | |} | ||
===Metal Accumulation=== | ===Metal Accumulation=== | ||
| + | |||
| + | *'''Check PCR on SmtA and GST-SmtA on gel''' | ||
| + | **Run 20uL (+ 1:6 dye) on 1% gel | ||
| + | **Contents: | ||
| + | ::2=1kb marker | ||
| + | ::4=GST-SmtA PCR product 1 --> expected size: 1400bp | ||
| + | ::6=SmtA PCR product 1 -->expected size: 660bp | ||
| + | ::8=GST-SmtA PCR product SmtA -->expected size: 660bp | ||
| + | [[Image:F102471 2009-08-06 SmtA pcr.JPG|gel]] | ||
| + | **No bands of expected size were found.. So PCR will be redone tomorrow. | ||
===Vectors=== | ===Vectors=== | ||
==Dry== | ==Dry== | ||
| + | Jasper worked on the equilibrium equations for arsenic transport. It proved a little difficult to derive an equation that does not depend on the extracellular concentration, that is, it appeared to show a bifurcation point (which doesn't seem very likely, although not completely impossible), if the (maximum) export rate becomes very high there would appear to be two solutions, neither of which can be ruled out very easily. This could use more analysis, but for the moment the derivation is commented out. | ||
{{Team:Groningen/Notebook/Day/Footer}} | {{Team:Groningen/Notebook/Day/Footer}} | ||
Latest revision as of 14:08, 7 August 2009
[http://2009.igem.org/Team:Groningen http://2009.igem.org/wiki/images/f/f1/Igemhomelogo.png]
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Wet
Ideas to improve ligation
- Blunt end ligation of vector with PCR fragment, use Klenow polymerase to blunt end the vector.
- Dephosphorylation of the vector via CAP.
- Do not (preferably) clean up PCR product from gel, but use ~1uL for checking on gel and clean the rest with a PCR cleanup kit.
- Use conventional restriction enzymes for digestion of the insert and vector, as these can be heat-inactivated.
- Increase the amount of insert from 1:3 to 1:6 or 1:10.
- Digest insert with XbaI and SpeI, these give compatible overhangs but these sites are closer to the gene (and have longer spacer).
GVP Cluster
Over Night Cultures
Over night cultures in 5mL LB-amp50 or LB-amp25/-chloramph100 medium were prepared from the following colonies:
- → E.coli TOP10 pSB1AC3-high const. promoter - GVP (amp.) (2x)
- → E.coli TOP10 pSB1AC3-med. const. promoter - GVP (amp.) (2x)
- → E.coli TOP10 pSB1AC3-low const. promoter - GVP (amp.) (2x)
- → E.coli TOP10 pSB1AC3-high const. promoter - GVP (amp./chl)
- → E.coli TOP10 pSB1AC3-med. const. promoter - GVP (amp./chl)
- → E.coli TOP10 pSB1AC3-low const. promoter - GVP (amp./chl)
- → E.coli TOP10 (oud) pSB1AC3-med. const. promoter - GVP (amp./chl)
and put in the 37°C waterbath at 200 rpm.
- → Note: The concentration of Chloramphe. antibiotics can be lower because crystalisation had occured during storage, vortexing did not dissolve the crystals completely!
Plasmid Purification
Plasmid isolation was performed on the cultures of E.coli TOP10 containing plasmids [http://partsregistry.org/wiki/index.php?title=Part:pSB3K3 pSB3K3] with high, medium and low constitutive promoters and [http://partsregistry.org/wiki/index.php?title=Part:BBa_I750016 BBa_I750016] 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 20μL MQ and stored in the fridge
Results: metal sensitive promoters in front of GVP
If the construct is correct one band at ~8kb should be visible. If the construct is incorrect one band at ~8kb and another at ~1.5kb should be visible. All plasmids have been cut with EcoRI and XbaI.
.JPG)
| 1) 1kb ladder | 10) 1kb ladder |
| 2) CueO- (1* concentrated) | 11) ZntR- (1* concentrated, diluted promoter) |
| 3) ZntR- (1* concentrated) | 12) ArsR- (1* concentrated, diluted promoter) |
| 4) ArsR- (1* concentrated) | 13) CueO- (9* concentrated, diluted promoter) |
| 5) CueO- (9* concentrated) | 14) ZntR- (9* concentrated, diluted promoter) |
| 6) ZntR- (9* concentrated) | 15) ArsR- (9* concentrated, diluted promoter) |
| 7) ArsR- (9* concentrated) | 16 GVP vector (cut with EcoRI and XbaI) |
| 8) CueO- (1* concentrated, diluted promoter) | 17) GVP vector (not digested) |
| 9) 1kb ladder | 18) 1kb ladder |
The 1 and 9 times concentration refers to the amount of cells used to plate. Diluted promoter refers to the amount of promoter DNA used in the ligation (a hundred times less used than undiluted).
Transporters
GlpF
Checking the previous ligation mixes by restriction with HaeII. HaeII cuts once in the psB1AC3 vector and once in the GlpF insert, so correct ligation results in two bands (2880 and 1019) if not a single band of 3055 is expected. HaeII cuts also twice in the ArsRMBP fusion (once in ArsR, once in MBP) and once in the psB1Ac3 vector so if the ArsRMBP insert is ligated correctly into the psB1Ac3 vector three bands are expected (507, 1068,2996) if not again a single band of 3055 is expected.
| 1 | 30/07/09 | 15uL | GlpF cut with XbaI,PstI | AC3 cut with EcoRI,SpeI |
| 2 | 31/07/09 | 20uL | GlpF cut with EcoRI,SpeI | AC3 cut with EcoRI,SpeI |
| 3 | 05/08/09 | 15uL | GlpF cut with XbaI,PstI | AC3 cut with XbaI,PstI |
| 4 | 05/08/09 | 15uL | ArsRMBP fusion cut with XbaI,PstI | AC3 cut with XbaI,PstI |
| Restriction mix | ||
| 8uL | ligation | → 37° 1.5h → 80° 20 min |
| 1uL | Tango | |
| 1uL | HaeII |
PCR GlpF
Redone PCR 1 and PCR 2
|
|
|
Metal Accumulation
- Check PCR on SmtA and GST-SmtA on gel
- Run 20uL (+ 1:6 dye) on 1% gel
- Contents:
- 2=1kb marker
- 4=GST-SmtA PCR product 1 --> expected size: 1400bp
- 6=SmtA PCR product 1 -->expected size: 660bp
- 8=GST-SmtA PCR product SmtA -->expected size: 660bp
- No bands of expected size were found.. So PCR will be redone tomorrow.
Vectors
Dry
Jasper worked on the equilibrium equations for arsenic transport. It proved a little difficult to derive an equation that does not depend on the extracellular concentration, that is, it appeared to show a bifurcation point (which doesn't seem very likely, although not completely impossible), if the (maximum) export rate becomes very high there would appear to be two solutions, neither of which can be ruled out very easily. This could use more analysis, but for the moment the derivation is commented out.
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