Team:Groningen/Project Plan/Construction

This is the iteration plan for the Construction iteration(s). See the UPEDU artifact description for information on what this is and what it contains (including a template).

Milestone
These are the major milestone objectives for the Construction phase :


 * Our wonderful buoyant, metal filtering bacteria should be alive and kicking!
 * (?) The lab/plan cloning strategy should be fully worked out at the end of the phase to reflect our final decisions on which parts/protocols/procedures/? to use. (I think this roughly corresponds to the Implementation Model in UPEDU. --Jaspervdg 11:09, 23 June 2009 (UTC))
 * The Iteration plan for the transition phase completed and reviewed.
 * (?) Design Model (and all constituent artifacts) updated with new design elements identified during the completion of all requirements. (I think this can roughly be seen as fleshing out all the device designs and parts, or in other words: making sure The Project is up-to-date. --Jaspervdg 11:09, 23 June 2009 (UTC))
 * More?

Resources

 * Note that we're opting to do this for the entire phase instead of each iteration as in UPEDU as this seems more natural (our needs change little over the course of one phase). --Jaspervdg 11:01, 23 June 2009 (UTC)

[Resources needed for the phase — material, human, financial, and so on.]

Construction 1
Overall objective:
 * The basic model should be ready
 * The seperate part of the labwork should be known and working or almost working
 * modellers should know which parameters they need so the labworkers can prepare providing these
 * The tickets and hostel should be booked

Week 27
June 30 - July 5 :


 * Analyst
 * Configuration Manager
 * Designer
 * Implementer
 * Have genes as BioBricks in the vector, together with all DNA elements needed for expression (RBS, term etc).
 * Work on the final parts should have been started.
 * Try to find protocols for the characterization, to check feasibility.
 * Keep record of the labwork on the Notebook site
 * GVP (Michael and Paul)
 * Test insert length of gvp cluster (E-genR-X-RBS-PART-S-P in vector BBa_J61035, Figure 1)
 * (Needed?) Silence restrictionsites in gvp cluster by PCR (BamHI, XhoI, BglII --> used in other BBa standard assemblies)and remove repeat in gvpL.
 * Add the terminator to the construct
 * If needed add promoter (constitutive promoter --> BBa_J23119 ) to do initial phenotype testing.
 * Metal transporters (Jolanda, SJ)
 * Transform E. coli with construct (HmtA first, get GlpF either by ordering or by genome PCR. Then order primers + pre/suffix and RBS in prefix).
 * Test insert length.
 * Silence restrictionsites in HtmA by PCR (2x PstI) and add BioBrick pre/suffix.
 * Clone the terminator in to the construct.
 * Metal accumulation (Wilfred, Nienke)
 * Decide which Metallothioneins will be used.
 * Transform E. coli with construct / synthetic gene.
 * Test insert length.
 * (If needed) Silence restrictionsites in gvp cluster by PCR.
 * Add BioBrick pre/suffix by PCR.
 * Clone the terminator in to the construct.
 * Vectors (Sven, Frans (Modelling aswell?)):
 * Transform E. coli TOP10 with pSB3K3 and E. coli DB3.1 with pSB1AC3.
 * pSB3K3 contains a p15A ORI and Kan resistance.
 * pSB1AC3 contains Amp + Cam resistance markers, a ccdB death gene and a pMB1 ORI.
 * Add different promoters to the vectors:
 * Inducible are: pBAD-AraC BBa_I0500 in pSB2K3 and the Lac promoter BBa_R0010 in pSB1A2.
 * Constitutive are: High expected expression yield, BBa_J23110 and Low expected expression yield, Lac promoter with mutated operator BBa_K119000 . For these promoter sequences are too small, oligo's have to be ordered + BBa pre/suffix !!without RBS!!
 * Metal sentitive are possibly copA BBa_J15502 (order oligo's) and ??
 * Clone in such a way that they are nicely -10 / -30 in front of the start codon.
 * Test functionality of the promoters by cloning GFP + RBS + Terminator behind the promoter (BioBrick??).
 * Integrator
 * Modeller
 * Project Manager
 * Have both modellers and labworkers up and running.
 * Labworkers have choosen the parts and tested those. The bouyancy, metal intake and accumulation should be tested and working seperatly.
 * Have a start-up model and make sure the labworkers know which parameters the modellers need and can or already have provided these.
 * Have choosen a tester, a "testplan" should have been made, the necessary equipment should be at least ordered.
 * the tickets and hostel should be booked, so plans for jamboree or thereafter should be known roughly.
 * Public Relations Officer
 * Get in contact with papers, magazines and other media for possible publications.
 * Keep in contact with other teams on progress during the summer.
 * Tester

Week 28
July 6 - July 12 :


 * Analyst
 * Configuration Manager
 * Designer
 * Implementer
 * Integrator
 * Modeller
 * ✅ Make a detailed list of things that should be measured in relation to buoyancy, indicating how well a guess we already have. For example (pretty much everything we'd love to have as a time series):
 * Densities (of cells).
 * Volume fraction of gas vesicles (needs to be determined how best to estimate this), to check the relationship between the density of the rest of the cell and the presence of gas vesicles.
 * (Relative) promotor strengths? (We should be able to relate this to gvp/transporter expression in some way!)
 * ✅ Reality check for buoyancy with arsenic.
 * Model accumulation of arsenic in E. coli.
 * ✅ How well does ArsR(+fusion protein) bind to its binding site.
 * How well does ArsR(+fusion protein) bind to arsenic.
 * ✅ Reaction rate(s) for binding, unbinding, production and degradation of ArsR(+fusion protein)?
 * Project Manager
 * Public Relations Officer
 * Tester

Week 29
July 13 - July 19 :

GVP (1pers):
 * Analyst
 * Configuration Manager
 * Designer
 * Implementer
 * Place three different constituive promotors with low, medium, and high activity in front of GVP-cluster
 * → ✅ cutting the vector with promotor sequence resulted in a fragment of about 50bp and was difficult to locate on 1% agarose gel
 * → ✅ gel purification of cut vector with GVP resulted in such a low concentration (4.3 ng/μL), it was not usable in the next step (ligation)
 * → ✅ next option is cutting out GVP-cluster, and ligate it into the promotor vector (other way around).

The cutting and ligation was one of the more serious issues of Melbourne 2007 team, and might give us the same problems to keep in mind
 * → ✅ check if transformation/ligation has worked (failed)
 * Isolate new plasmids of GVP and the three promotors to continu work
 * → ✅ grow new cultures of each from the glycerol stocks
 * → ✅ determine concentration with nanodrop
 * Create (and test) variants of GVP vector with/without Ori+resistance between the E and X sites???
 * → get in contact with Kloosterman for protocols for working with small fragments
 * → ✅ isolate cut promotors from gel (failed)

Metal transporters (2 pers):
 * PCR HmtA???
 * PCR GlpF???

Metal accumulation (2 pers):
 * ArsR fusion???

Vectors (1pers):
 * Two variants of promotor + RBS (one with RBS from genome and one with RBS from GVP).???

All (8pers):
 * List for -80°C and -20°C storage
 * → ✅ list for -80°C storage can be found here, but needs finishing
 * → ✅ storage in -20°C is kept up to data by the persons working in the lab, and excel sheats are made for on the door
 * Cloning strategy (make a plan up to the final assembly)
 * → what are the target vectors going to be, and which genes are going to be grouped together
 * → what is still missing
 * → are there options to be investigated


 * Integrator
 * Modeller
 * ✅ Make a detailed list of things that should be measured, indicating how well a guess we already have. For example (pretty much everything we'd love to have as a time series):
 * Concentrations (of metals).
 * Concentrations of bound metals vs. unbound metals in cells?
 * (Relative) promotor strengths? (We should be able to relate this to gvp/transporter expression in some way!)
 * Model import/export of arsenic in E. coli (use surface area per volume of cells + number of transporters per surface as measure?).
 * ✅ Find diffusion speed (if nonzero). (Most likely does not diffuse through the cell membrane.)
 * ✅ What transporters in and out of the cell are there in our E. coli.
 * How much transporters in and out of the cell are there in our E. coli.
 * How much are we adding?
 * ✅ "Reaction" rate(s) for import (GlpF).
 * ✅ "Reaction" rate(s) for export (ArsB).
 * Project Manager
 * Public Relations Officer
 * Write press release


 * Tester

Construction 2
Overall objective:

Tasks per role :


 * Analyst
 * Configuration Manager
 * Designer
 * Implementer and Tester
 * GVP (1 pers):
 * Initial testing of phenotype, growth rates E. coli
 * Clone gvp from BBa_J61035 to the high / low copy number vector
 * (If the vectors do not already contain one) Clone gvp in vector with different promoters; high, moderate, low expression.
 * Metal transporters (2 pers):
 * Initial testing of phenotype, growth rates E. coli.
 * Clone HtmA to the high / low copy number vector.
 * (If the vectors do not already contain one) Clone gvp in vector with different promoters; high, moderate, low expression (see GVP). --> do not use constitutive promoter before a combined construct with a metal accumulator has been made!
 * Metal accumulation (2 pers):
 * Initial testing of phenotype, growth rates E. coli.
 * Clone the gene to the high / low copy number vector.
 * (If the vectors do not already contain one) Clone gvp in vector with different promoters; high, moderate, low expression (see GVP).
 * Vectors (1pers):
 * Add different promoters to the vectors--> in such a way that they are nicely -10 / -30 in front of the start codon.
 * Test functionality of the promoters by cloning GFP + RBS + Terminator behind the promoter.
 * Integrator
 * Modeller
 * Project Manager
 * Public Relations Officer

Week 30
July 20 - July 26 :

GVP (1 pers):
 * Analyst
 * Configuration Manager
 * Designer
 * Implementer
 * Place promotors BBa_J23109, BBa_J23100, and BBa_J23106 in front of GVP in either the plasmid of the promotor or the GVP-cluster
 * → make a cloning strategy for best assembly, and get familiar with procedure
 * Gel purification has to be obtimised for the standard plasmid purification kit, or a specialised kit has to be ordered
 * Primers/Oligo's for three metal induced promotors should arive
 * → select vector to ligate fragment in for boibrick storage
 * → ligate parts directly infront of GVP-cluster
 * Expression of GVP genes in E.coli TOP10 cells test strategy
 * → start can be test in 50mL tubes and compare "floating" of cells with and without plasmid
 * → the test tube of 1 meter in lenght can be a good follow up test if floatation is observed
 * Clone GVP-cluster from vector BBa_J61035 to the high / low copy number vector
 * → deside together with persons from vector group which vectors they have selected and prepared
 * → clone GVP-cluster + RBS + Promotor (Terminator) in new vectors
 * Improving GVP biobrick compared to Melbourne part
 * → how can the insert of 40bp repeat be removed from gvpL?
 * → remove the RBS which is infront of gvp-cluster to add own RBS
 * → the original DNA cluster, including gvpA, should be obtained
 * → make primers for gvpA and obtain the gene as biobrick (can be used to replace gvpB and see its affect)
 * Make contact with other iGEM teams with related projects
 * → formulate questions for Melbourne iGEM 2007 team, and get in contact with their supervisor(s): Paul Gooley and Heung-Chin Cheng
 * → Newcastle University is working on metal uptake and storage by B. subtilis, try to get in contact

Metal transporters (2 pers):


 * PCR the HmtA gene with "old" primers
 * → test forward HmtA primer
 * → Order new HmtA primers in order to remove the extra EcoR1 site
 * If GlpF primers are in, PCR the GlpF gene, from the genome of e.coli,in two PCR reactions.
 * → first PCR with forward and mutation reverse and second the reverse with the mutation forward.

Metal accumulation (2 pers):

Vectors (1pers):

All (8pers):
 * Cloning strategy (make a plan up to the final assembly)
 * → what are the target vectors going to be, and which genes are going to be grouped together
 * → what is still missing
 * → are there options to be investigated


 * Integrator
 * Modeller
 * Project Manager
 * Public Relations Officer
 * Tester

Week 31
July 27 - August 2 :


 * Analyst
 * Configuration Manager
 * Designer
 * Implementer
 * GVP (1 pers):
 * Place promotors BBa_J23109, BBa_J23100, and BBa_J23106 in front of GVP in either the plasmid of the promotor or the GVP-cluster
 * make a cloning strategy for best assembly, and get familiar with procedure
 * Gel purification has to be obtimised for the standard plasmid purification kit, or a specialised kit has to be ordered
 * Primers/Oligo's for three metal induced promotors should arive
 * select vector to ligate fragment in for boibrick storage
 * ligate parts directly infront of GVP-cluster
 * Expression of GVP genes in E.coli TOP10 cells test strategy
 * start can be test in 50mL tubes and compare "floating" of cells with and without plasmid
 * the test tube of 1 meter in lenght can be a good follow up test if floatation is observed
 * Clone GVP-cluster from vector BBa_J61035 to the high / low copy number vector
 * deside together with persons from vector group which vectors they have selected and prepared
 * clone GVP-cluster + RBS + Promotor (Terminator) in new vectors
 * Improving GVP biobrick compared to Melbourne part
 * how can the insert of 40bp repeat be removed from gvpL?
 * the original DNA cluster, including gvpA, should be obtained
 * make primers for gvpA and obtain the gene as biobrick (can be used to replace gvpB and see its affect)
 * Make contact with other iGEM teams with related projects
 * formulate questions for Melbourne iGEM 2007 team, and get in contact with their supervisor(s): Paul Gooley and Heung-Chin Cheng
 * Newcastle University is working on metal uptake and storage by B. subtilis, try to get in contact
 * Metal transporters (2 pers):
 * Metal accumulation (2 pers):
 * ✅ Transform E. coli with SmtA and SmtA-GST plasmids.
 * Check SmtA and SmtA-GST constructs.
 * Put SmtA, MymT, fMT, ArsR on SandBox.
 * Vectors (1pers):
 * ✅Check pSB3K3 and pSB1AC3 + constitutive promoters
 * Integrator
 * Modeller
 * ✅ Get all data we use from Google spreadsheets to tables on the Wiki.
 * Plan for experiment for determining Kd values (for ArsR with arsenic/promoter).
 * Project Manager
 * Public Relations Officer
 * Tester

Week 32
August 3 - August 9 :

GVP (1 pers):
 * Analyst
 * Configuration Manager
 * Designer
 * Implementer
 * Place promotors BBa_J23109, BBa_J23100, and BBa_J23106 in front of GVP in either the plasmid of the promotor or the GVP-cluster
 * → ✅ make a cloning strategy for best assembly, and get familiar with procedure
 * Gel purification has to be obtimised for the standard plasmid purification kit, or a specialised kit has to be ordered ✅
 * Primers/Oligo's for three metal induced promotors should arive
 * → ✅ select vector to ligate fragment in for boibrick storage (J61002)
 * → ✅ ligate parts directly infront of GVP-cluster
 * Expression of GVP genes in E.coli TOP10 cells test strategy
 * → ✅ start can be test in 50mL tubes and compare "floating" of cells with and without plasmid
 * → the test tube of 1 meter in lenght can be a good follow up test if floatation is observed
 * Clone GVP-cluster from vector BBa_J61035 to the high / low copy number vector
 * → ✅ deside together with persons from vector group which vectors they have selected and prepared
 * → ✅ clone GVP-cluster + RBS + Promotor (Terminator) in new vectors (into 4 vectors)
 * Improving GVP biobrick compared to Melbourne part
 * → ✅ how can the insert of 40bp repeat be removed from gvpL (strategy desided)
 * → remove the RBS which is infront of gvp-cluster to add own RBS
 * → the original DNA cluster, including gvpA, should be obtained
 * → make primers for gvpA and obtain the gene as biobrick (can be used to replace gvpB and see its affect)
 * Make contact with other iGEM teams with related projects
 * → formulate questions for Melbourne iGEM 2007 team, and get in contact with their supervisor(s): Paul Gooley and Heung-Chin Cheng
 * → Newcastle University is working on metal uptake and storage by B. subtilis, try to get in contact


 * Metal Accumulation
 * ✅ Check SmtA and SmtA-GST constructs.
 * Put SmtA, MymT, fMT, ArsR on SandBox.
 * Amplify SmtA and SmtA-GST by PCR
 * Transform E. coli with pGB68-mymT
 * Vectors
 * Construct pSB1AC3-Med constitutive promoter with RFP
 * Check expression of RFP for pSB3K3-HML, pSB1AC3-HML
 * Construct pSB3K3, pSB1AC3 with metal sensitive promoters
 * Integrator
 * Modeller
 * Reduce dependency on dojo? (Use it just for graphing.)
 * ✅ Plans for other experiments?
 * ✅ Jasper: Plan uptake experiment with Nienke.
 * ✅ Annelies: Plan experiment to determine density of unmodified E. coli with Frans(/Sven). (In order to check the feasibility.)
 * Annelies: Help with experiment to determine density of unmodified E. coli with Frans(/Sven). (In order to check the feasibility.)
 * Dissociation constants/production rates etc. are not going to happen for now...
 * Characterizing our metal sensitive promoter(s) should be possible, but not doing that for now.
 * ✅ Figure out what experimental results from papers we could try to reproduce using our models?
 * Figure 3A from Kostal2004.
 * Figure 1B from Singh2008.
 * ??? Figure 2 from Dey1995.
 * Project Manager
 * Public Relations Officer
 * Tester

Construction 3
Overall objective:

Tasks per role :


 * Analyst
 * Configuration Manager
 * Designer
 * Implementer and Tester
 * GVP (1 pers):
 * Test phenotype, growth rates E. coli again.
 * Transform E. coli with both vectors
 * Check compatibility of gvp expression and metal transport / accumulation system expression in E. coli by transforming the organism with both vectors and select on two antibiotics (compatible selection markers and promoters)
 * Make final buoyancy tests.
 * Metal transporters (2 pers):
 * Test phenotype, growth rates E. coli again.
 * Transform E. coli with both vectors.
 * Check compatibility of gvp expression and metal transport / accumulation system expression in E. coli by transforming the organism with both vectors and select on two antibiotics (compatible selection markers and promoters)
 * Make final metal importing tests.
 * Metal accumulation (2 pers):
 * Test phenotype, growth rates E. coli again.
 * Transform E. coli with both vectors.
 * Check compatibility of gvp expression and metal transport / accumulation system expression in E. coli by transforming the organism with both vectors and select on two antibiotics (compatible selection markers and promoters)
 * Make final metal accumulation tests.
 * Integrator
 * Modeller
 * Project Manager
 * Public Relations Officer

Week 33
August 10 - August 16 :


 * Analyst
 * Configuration Manager
 * Designer
 * Implementer
 * GVP:
 * Check Bouyancy phenotype of E. coli TOP10 + pNL29 (GVP) and WT.
 * Metal transporters (2 pers):
 * Metal accumulation (2 pers):
 * ✅ Check pGB68-mymT construct
 * ✅ Amplify MymT by PCR, ligate in pSB1AC3
 * PCR / Put SmtA in pSB1AC3
 * Check Arsenic uptake/transport in E. coli TOP10 WT
 * Vectors (1pers):
 * Integrator
 * Modeller
 * Clean-up modelling sections (references where references are due, check consistency, extra diagrams/explanation where needed), partially based on Wilfred's feedback
 * Metal accumulation: Annelies
 * Metal transport: ???
 * Gas vesicle: ???
 * ✅ Adjust ArsB model to incorporate regulation by ArsR.
 * Project Manager
 * Public Relations Officer
 * Tester

Week 34
August 17 - August 23 :

GVP (1 pers):
 * Analyst
 * Configuration Manager
 * Designer
 * Implementer
 * Place metal promotors in plasmid J61002 for expression test with RFP and submission to the registry.
 * Expression of GVP genes in E.coli TOP10 cells test strategy
 * → ✅ start can be test in 50mL tubes and compare "floating" of cells with and without plasmid
 * → the test tube of 1 meter in lenght can be a good follow up test if floatation is observed
 * Clone GVP-cluster from vector BBa_J61035 to the high / low copy number vector
 * → ✅ deside together with persons from vector group which vectors they have selected and prepared
 * → clone GVP-cluster + RBS + Promotor (Terminator) in new vectors (into 4 vectors)(has to be finished)
 * Improving GVP biobrick compared to Melbourne part
 * → remove insert of 40bp repeat from gvpL (strategy desided)
 * → remove the RBS which is infront of gvp-cluster to add own RBS
 * → the original DNA cluster, including gvpA, should be obtained
 * → make primers for gvpA and obtain the gene as biobrick (can be used to replace gvpB and see its affect)
 * Make contact with other iGEM teams with related projects
 * → formulate questions for Melbourne iGEM 2007 team, and get in contact with their supervisor(s): Paul Gooley and Heung-Chin Cheng
 * → Newcastle University is working on metal uptake and storage by B. subtilis, try to get in contact


 * Metal Accumulation
 * ✅ Check SmtA and SmtA-GST constructs.
 * ✅ Check pSB1AC3-MymT transformants from last week.
 * ✅ Put SmtA, MymT, fMT, ArsR on SandBox.
 * ✅ Amplify SmtA (SmtA-GST), fMT and MymT by PCR
 * ✅ Transform E. coli with pSB1AC3-mymT
 * Check As uptake by resting E. coli WT cells
 * Vectors
 * Integrator
 * Modeller
 * (Post-poned) Animated interface for model (computations need not be correct, just checking the feasibility at this time).
 * Arsenic floating through a solution
 * Cells floating through the same solution
 * Somehow either sense collisions (and decide whether arsenic crosses the boundary or not) or avoid unwanted collisions?
 * Comparison between our model and experimental results on the Wiki.
 * Project Manager
 * Public Relations Officer
 * Tester
 * Tester

Week 35
August 24 - August 30 :


 * Analyst
 * Configuration Manager
 * Designer
 * Implementer
 * Integrator
 * Modeller
 * (Post-poned) Working graphical interface for model.
 * ✅ Modelling page cleaned-up and made more "user-friendly" (nice images, clear explanations, etc.)
 * Project Manager
 * Public Relations Officer
 * Tester