Team:Groningen/Project/Lab plan

From 2009.igem.org

(Difference between revisions)
(To do list)
(Replacing page with '{{Team:Groningen/Header}} ='''Lab plan:'''= For Basic Cloning strategy and final products see [https://2009.igem.org/Team:Groningen/Project The Project] =='''Wk1 / Wk2'''== Fo...')
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{{Team:Groningen/Header}}
{{Team:Groningen/Header}}
='''Lab plan:'''=
='''Lab plan:'''=
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For Basic Cloning strategy and final products see [https://2009.igem.org/Team:Groningen/Project The Project]
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# Clone gvp from BioBrick (BBa_I750016) in vector with inducible promoter (e.g. PAra)
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=='''Wk1 / Wk2'''==
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# Clone a metal ion transporter (CitM and HtmA are planned)
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For weekly of the lab work planning see Notebook --> Mondays
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# Clone a metal accumulating protein.
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# Clone a metal sensitive promoter in front of the gvp cluster.
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# Clone the metal accumulating protein and metal transporter in one vector, possibly on a synthetic operon
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# Transform both vectors in one ''E. coli'' strain
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=='''Wk1'''==
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==='''Gvp Cluster'''===
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[[Image:BBa_J61035.jpg|thumb| Figure 1: BBa_61035]]
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*BBa_I750016 is a construct: E-genR-X-RBS-PART-S-P in vector BBa_J61035.
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*Possibly the AmpR and ColE1 are also still present on the vector, as the part was last checked in 2008 and contained a Amp resistance marker…
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*The part is present in the microtiter plate: Kit Plate 2, Well 11A
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*Quality control was okay, but plasmid length was different than expected. --> should be checked by us.
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*Plan:
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**Get chemically competent ''E. coli''
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**Dilute dry DNA of gvp with 15ul diH2O
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**Transform to ''E. coli'' --> see protocol
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**Plate on LB-A plates with Amp or Gentamicin selection (50/50)
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**Use positive and negative control
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**Grow o/n @ 37 degrees
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**Pick single colony and grow o/n in 5ml LB + correct antibiotic
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***Make glycerol stocks of the o/n culture
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***Do DNA isolation
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***Check the vectors insert by restriction analysis --> ''Pst''I and ''Xba''I (would give 1 band of ~ 3539bp and 1 of 6064bp)
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*Design primers to get rid of restrictionsites
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==='''CitM'''===
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*SJ transformed ''E. coli'' with pWSK29, a low copy nr vector containing CitM (according to B. Krom this is the best vector for CitM)
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*Get plasmid and possibly the plate with ''E. coli'' transformants.
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**Check expression of CitM while growing on citrate medium with Amp, induce with IPTG
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*Check gene for restriction sites and design primers to get rid of restriction sites.
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*Design primers to introduce a pre/suffix --> ask B. Krom whether this would be feasible
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==='''HtmA'''===
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* On its way, but hopefully arrives next week
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==='''Accumulation of metal'''===
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*BioBrick (BBa_K129004) LamB + metal binding domain is not available
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*Find out whether it’s possible to only express the metal binding domain (6AA) and if so, order synthetic DNA with part with pre/suffix
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[[Image:PACYC-OmpA_BBa_K103017.png|thumb| Figure 2: BBa_K103017 a derivative of pACYC177]]
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==='''To do list'''===
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*Get CitM from the biological centre
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*Finish equipment list --> send to facility manager
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*Design primers
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*Read articles to find [https://2009.igem.org/Team:Groningen/Project modeling parameters] for the different sub-projects and to find testing-phenotype (e.g. metal inport) protocols
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*Start ordering materials like: media, restriction enzymes, kits
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*Talk with Profs for permission to use stuff from the different groups in Haren
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*Check compatibility of different plasmids / promoters / replicons in ''E. coli''--> On Fermentas site about  [http://www.fermentas.com/techinfo/nucleicacids/mappuc1819.htm pUC18, pUC19] and about [http://www.fermentas.com/techinfo/nucleicacids/mappacyc184.htm pACYC184]
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** ORI's pMB1, ColE1 are incompatible, but p15A is compatible with ColE1 or pMB1 (pWSK29 is a pBluescript derivative with pMB1 ORI and AmpR)
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** Nis/subtilin promoters cannot be used simultaneously
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*Make a plan for July, concerning:
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**Vector --> how to get the pBS3K1 empty, what kind of vector is pWSK29 (Frans)
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***[https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega-A-alpha pACYC177] vector with kan and p15a (Figure 2)
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****Cut it with PstI and XbaI, run on gel and isolate correct fragment
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****Insert oligo with PstI-BioBrick Prefix-promoter-RBS-terminator(?)-BioBrick Suffix-XbaI
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**Primers --> how to remove restrictionsites in gvp, possibly in CitM / HtmA (PstI)
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**Promoters (Frans, Michael)
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***High/moderate/low expression upon induction or constitutive
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***Metal sensitive promotor
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*[http://partsregistry.org/Featured_Parts:CloningFacilitationTools cloning facilitation tools]
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=='''Wk 2:'''==
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*Get everything in the lab
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*Have BioBricks checked on correct insert
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*Have primers ordered
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=='''July:'''==
=='''July:'''==
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For detailed planning of the labwork for different groups see the Construction phases in [https://2009.igem.org/Team:Groningen/Project_Plan Project Plan] for the functions Implementer and Tester (mostly combined for lab work)
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GVP (2 people)
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#Test insert length of gvp cluster
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#Clone gvp from BBa_J61035 to another vector with p15A vector (pBS3K1)
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#Clone gvp in vector with different promoters; high, moderate, low expression…
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#Silence restriction sites in gvp cluster by PCR (which?)
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#Test phenotype, growth rates E. coli --> which of the promoters is the best one?
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#Check compatibility of gvp expression and CitM expression in E. coli by transforming the organism with both vectors and select on two antibiotics (compatible selection markers and promoters)
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Metal transporters (3 people)
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*CitM
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#Transform E. coli with pWSK29
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#Test phenotype, expression, check growth rate while expressing CitM
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Metal accumulation (3 people)
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Revision as of 21:16, 23 June 2009

[http://2009.igem.org/Team:Groningen http://2009.igem.org/wiki/images/f/f1/Igemhomelogo.png]

Lab plan:

For Basic Cloning strategy and final products see The Project

Wk1 / Wk2

For weekly of the lab work planning see Notebook --> Mondays

July:

For detailed planning of the labwork for different groups see the Construction phases in Project Plan for the functions Implementer and Tester (mostly combined for lab work)