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