"
Team:Groningen/Project
From 2009.igem.org
(Difference between revisions)
| Line 1: | Line 1: | ||
{{Team:Groningen/Project/Header}} | {{Team:Groningen/Project/Header}} | ||
| + | |||
| + | |||
| + | == For the lab work of our project, concerning the bouyant bacteria with its metal absorbing and accumulating "function", we hope to produce the following products by using our basic cloning strategy. | ||
| + | == | ||
| + | |||
| + | '''Final products:''' | ||
| + | * Plasmid with gvp cluster, regulated by different promoters. [BioBricks] | ||
| + | * (Several) plasmid(s) with a metal transporter, a metal accumulating protein and if needed a regulator protein for the metal sensitive promoter. [BioBricks] | ||
| + | |||
| + | |||
| + | '''Basic Cloning Strategy:''' | ||
| + | # Transform E. coli with gvp (BBa_I750016), a metal ion transporter (HmtA and GlpF) and accumulation proteins. | ||
| + | ## Test expression / phenotype of separate proteins (if possible in the vectors they are supplied in). | ||
| + | # Put both systems (gvp and metal import) on a high and low copy number to prevent that the plasmid / expression of one of the systems is not compatible with the other. E.g. use pSB3K3 with p15A, Kan resistance and pSB1AC3 containing Amp + Cam and a pMB1 ori. | ||
| + | ## The metal transporter and accumulation protein should be cloned in parallel. | ||
| + | ##Then clone them in one vector (in parallel for different systems for Cu, Zn, As, (Hg)). If possible on a synthetic operon. | ||
| + | # PCR the restriction sites out and add BioBrick pre- and suffix. | ||
| + | ##Primers should be ordered for HtmA | ||
| + | # Add a RBS (BBa_B0034) and a terminator (BBa_B0014). | ||
| + | ## For gvp the RBS is included in the construct. | ||
| + | # In parallel clone metal sensitive promoters in front of a fluorescent protein (GFP) and in front of the gvp cluster. | ||
| + | # In parallel clone different promoters (inducible like Para or Plac, constitutive with expected high and low expression yield) in front of the two systems. | ||
| + | # Then try to get both systems in one E. coli strain, test different possibilities with the high + low copy nr vectors. | ||
| + | |||
'''Planning and requirements:''' | '''Planning and requirements:''' | ||
Revision as of 19:39, 23 June 2009
 | The Project
|
|---|
== For the lab work of our project, concerning the bouyant bacteria with its metal absorbing and accumulating "function", we hope to produce the following products by using our basic cloning strategy.
==
Final products:
- Plasmid with gvp cluster, regulated by different promoters. [BioBricks]
- (Several) plasmid(s) with a metal transporter, a metal accumulating protein and if needed a regulator protein for the metal sensitive promoter. [BioBricks]
Basic Cloning Strategy:
- Transform E. coli with gvp (BBa_I750016), a metal ion transporter (HmtA and GlpF) and accumulation proteins.
- Test expression / phenotype of separate proteins (if possible in the vectors they are supplied in).
- Put both systems (gvp and metal import) on a high and low copy number to prevent that the plasmid / expression of one of the systems is not compatible with the other. E.g. use pSB3K3 with p15A, Kan resistance and pSB1AC3 containing Amp + Cam and a pMB1 ori.
- The metal transporter and accumulation protein should be cloned in parallel.
- Then clone them in one vector (in parallel for different systems for Cu, Zn, As, (Hg)). If possible on a synthetic operon.
- PCR the restriction sites out and add BioBrick pre- and suffix.
- Primers should be ordered for HtmA
- Add a RBS (BBa_B0034) and a terminator (BBa_B0014).
- For gvp the RBS is included in the construct.
- In parallel clone metal sensitive promoters in front of a fluorescent protein (GFP) and in front of the gvp cluster.
- In parallel clone different promoters (inducible like Para or Plac, constitutive with expected high and low expression yield) in front of the two systems.
- Then try to get both systems in one E. coli strain, test different possibilities with the high + low copy nr vectors.
Planning and requirements:
- Gasvesicle:
- Modelling:
- Buoyancy
- Permeability
- number of gasvesicles
- Where do the gasvesciles end, in hight.
- Mass of the bacteria (e.coli)
- How long it takes before it floates
- how long it take untill it is being expressed
- How long it will take untill there are enough gasvesicles
- how does it stay floating
- Buoyancy
- Lab
- GvP
- Cluster of biobricks
- Vector ordered from article
- first expression with constutatieve promotor, later with metal sensitive promotor
- Measurements
- Where are the bacteria, what are bac concentrations on a certain hight
- Making pictures on certain hight and compare with picture of known concentration
- Where are the bacteria, what are bac concentrations on a certain hight
- GvP
- Modelling:
- Metal importers
- Modelling
- Import speed
- Amount
- Max
- Lab
- HmtA
- Zn/Cu alone
- B-type ATPase (could be use if there is a ATP shortage?)
- CitM
- Divalent ions
- Citrate around
- Citrate can bind metals that are already bound.
- Measurements
- Transporter, on/off mechanism, up to what concentration (in the cell) does it still have metal uptake.
- Measure concentration of metal. difference between begin and end concentrations of metal outside the cell
- HmtA
- Modelling
- Promotors
- Modelling
- How do they react to metal, metal concentration vs expression of the protein after promotor (bv GFP)
- Lab
- Measuments
- Expression of the gene behind the promotor(GFP)
- Cu-promotor
- Arc promotor
- Measuments
- Modelling
- Accumulation
- Modelling
- Speed
- Metaliotheines concentration
- Lab
- Measurements
- Measure accumulation. By measuring before/after concentration metal with and without accumulation protein.
- Biobrick Bba_K129004
- Rest
- Measurements
- Modelling
Order of action
- Buoyancy
- Metal importation
- Accumulation
- Metal sensitive promotor
