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Team:Groningen/Notebook/14 August 2009
From 2009.igem.org
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===GVP Cluster=== | ===GVP Cluster=== | ||
| - | :→ {{ | + | :→ {{done}} make glycerol stocks for pSB1AC3-BBa_J23106-BBa_I750016 and pSB1AC3-BBa_J23109-BBa_I750016 (two o.n. cultures of each are grown) |
| - | :→ {{ | + | :→ {{done}} isolate plasmid J61002-BBa_J23100, J61002-BBa_J23106, and J61002-BBa_J23109 from o.n. cultures |
| - | :→ {{ | + | :→ {{done}} make a planning for next week |
'''Plates''' | '''Plates''' | ||
| Line 13: | Line 13: | ||
Showed single colony growth on plates with pSB3K3-H/L-GVP plasmids, and were stored in the fridge for future preculture growth. | Showed single colony growth on plates with pSB3K3-H/L-GVP plasmids, and were stored in the fridge for future preculture growth. | ||
| - | :→ The plates with pSB1AC3-H-GVP plasmid showed no growth, and were stored at | + | :→ The plates with pSB1AC3-H-GVP plasmid showed no growth, and were stored at 37°C for an additional 4 hours to be sure. Still no growth after addition incubation at 37°C, can be due to high energy cost of vesicle production resulting in the death of the cells! |
'''Over Night Cultures''' | '''Over Night Cultures''' | ||
| - | :→ All cultures showed growth, and can be used for plasmid isolation and glycerol stocks. | + | :→ All cultures showed growth, and can be used for plasmid isolation and glycerol stocks. From each culture 800uL was added to 300uL 87% glycerol and frozen in liquid nitrogen. Stocks were stored at position 21 to 24 in the -80°C fridge. |
[[Image:Plasmid Miniprep Kit Flow Protocol.gif|thumb|150px| www.sigmaaldrich.com]] | [[Image:Plasmid Miniprep Kit Flow Protocol.gif|thumb|150px| www.sigmaaldrich.com]] | ||
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* From each tube 8mL 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 8mL 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 | + | * Plasmids were eluted with 50μL MQ and stored in the fridge |
| + | |||
| + | '''Concentration''' | ||
| + | |||
| + | {|cellpadding="2" cellspacing="1" border="4" | ||
| + | |'''Plasmid''' | ||
| + | |'''Conc. ng/μL''' | ||
| + | |'''260/280 | ||
| + | |'''260/230 ''' | ||
| + | |'''-20 box (michael | ||
| + | |'''Restriction Control''' | ||
| + | |- | ||
| + | |BBa_J61002-BBa_J23100 (high+RFP) | ||
| + | |288.1 | ||
| + | |1.90 | ||
| + | |2.33 | ||
| + | |D-8 | ||
| + | |No | ||
| + | |- | ||
| + | |BBa_J61002-BBa_J23106 (med.+RFP) | ||
| + | |162.2 | ||
| + | |1.93 | ||
| + | |2.31 | ||
| + | |D-9 | ||
| + | |No | ||
| + | |- | ||
| + | |BBa_J61002-BBa_J23109 (low+RFP) | ||
| + | |114.2 | ||
| + | |1.93 | ||
| + | |2.28 | ||
| + | |D-10 | ||
| + | |No | ||
| + | |} | ||
===Transporters=== | ===Transporters=== | ||
| - | === | + | [[Image:F102471_2009-08-14_12hr_03min_PCR1_PCR2_Dynazyme_XbaI.jpg|thumb|PCR2, PCR1 Dynazyme XbaI]] |
| + | ====HmtA==== | ||
| + | After 2 weeks of PCR-ing plasmid isolations there is a product worth wile to send for sequencing. 4,5,6, and 8 | ||
| + | In the first 4 lanes PCR2 (261bp) is created from HmtA-AC3 plasmid with PstI 's left in place. In lane 5 till 8 PCR1 (1153bp)is made from HmtA-AC3 plasmid with PstI 's left in place to make mega primers to mutate the restriction sites. | ||
| + | |||
| + | In the lower left quarter is a PCR on HmtA-AC3 plasmid with PstI 's left in place with VF2 VR and failed. | ||
| + | The second lower quarter is a XbaI restriction control on plasmids 4,5,6 and 8 not completely digested but indicating the construct is correct in the way that there is a restriction site of the bio brick, meaning the EcoRI mutation got removed. | ||
| + | |||
| + | ===Metal Accumulation=== | ||
| + | *'''ligate MymT in pSB1AC3''' | ||
| + | **Purify mymT-RBS-pre/suff from gel using Roche Gel Extraction kit | ||
| + | **Elute in 25uL TE buffer (pH8) | ||
| + | **Digest mymT-RBS-pre/suff with EcoRI and Spe I | ||
| + | **Ligate with pSB1AC3/EcoRI-SpeI | ||
===Vectors=== | ===Vectors=== | ||
==Dry== | ==Dry== | ||
| + | Jasper changed [[Team:Groningen/Modelling/Arsenic|the model]] to take dimeric binding of ArsR into account and to ignore ArsD, as well as putting an image map on-line as the first step towards a clearer interface. The new model leads to a number of third order equations, which means that it is no longer feasible to give closed-form solutions, but it does seem perfectly feasible to compute the solutions numerically (as demonstrated by the updated calculator on the [[Team:Groningen/Project/Accumulation|accumulation page]]). | ||
{{Team:Groningen/Notebook/Day/Footer}} | {{Team:Groningen/Notebook/Day/Footer}} | ||
Latest revision as of 06:49, 17 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 make glycerol stocks for pSB1AC3-BBa_J23106-BBa_I750016 and pSB1AC3-BBa_J23109-BBa_I750016 (two o.n. cultures of each are grown)
- → DONE isolate plasmid J61002-BBa_J23100, J61002-BBa_J23106, and J61002-BBa_J23109 from o.n. cultures
- → DONE make a planning for next week
Plates
Showed single colony growth on plates with pSB3K3-H/L-GVP plasmids, and were stored in the fridge for future preculture growth.
- → The plates with pSB1AC3-H-GVP plasmid showed no growth, and were stored at 37°C for an additional 4 hours to be sure. Still no growth after addition incubation at 37°C, can be due to high energy cost of vesicle production resulting in the death of the cells!
Over Night Cultures
- → All cultures showed growth, and can be used for plasmid isolation and glycerol stocks. From each culture 800uL was added to 300uL 87% glycerol and frozen in liquid nitrogen. Stocks were stored at position 21 to 24 in the -80°C fridge.
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_J23100 high], [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23106 medium] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23109 low] constitutive promoters 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 8mL 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 50μL MQ and stored in the fridge
Concentration
| Plasmid | Conc. ng/μL | 260/280 | 260/230 | -20 box (michael | Restriction Control |
| BBa_J61002-BBa_J23100 (high+RFP) | 288.1 | 1.90 | 2.33 | D-8 | No |
| BBa_J61002-BBa_J23106 (med.+RFP) | 162.2 | 1.93 | 2.31 | D-9 | No |
| BBa_J61002-BBa_J23109 (low+RFP) | 114.2 | 1.93 | 2.28 | D-10 | No |
Transporters
HmtA
After 2 weeks of PCR-ing plasmid isolations there is a product worth wile to send for sequencing. 4,5,6, and 8
In the first 4 lanes PCR2 (261bp) is created from HmtA-AC3 plasmid with PstI 's left in place. In lane 5 till 8 PCR1 (1153bp)is made from HmtA-AC3 plasmid with PstI 's left in place to make mega primers to mutate the restriction sites.
In the lower left quarter is a PCR on HmtA-AC3 plasmid with PstI 's left in place with VF2 VR and failed. The second lower quarter is a XbaI restriction control on plasmids 4,5,6 and 8 not completely digested but indicating the construct is correct in the way that there is a restriction site of the bio brick, meaning the EcoRI mutation got removed.
Metal Accumulation
- ligate MymT in pSB1AC3
- Purify mymT-RBS-pre/suff from gel using Roche Gel Extraction kit
- Elute in 25uL TE buffer (pH8)
- Digest mymT-RBS-pre/suff with EcoRI and Spe I
- Ligate with pSB1AC3/EcoRI-SpeI
Vectors
Dry
Jasper changed the model to take dimeric binding of ArsR into account and to ignore ArsD, as well as putting an image map on-line as the first step towards a clearer interface. The new model leads to a number of third order equations, which means that it is no longer feasible to give closed-form solutions, but it does seem perfectly feasible to compute the solutions numerically (as demonstrated by the updated calculator on the accumulation page).
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