Team:Groningen/Project Plan/Construction

From 2009.igem.org

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(Week 29)
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* [[:Category:Team:Groningen/Roles/Designer|Designer]]
* [[:Category:Team:Groningen/Roles/Designer|Designer]]
* [[:Category:Team:Groningen/Roles/Implementer|Implementer]]
* [[:Category:Team:Groningen/Roles/Implementer|Implementer]]
-
** Create GVP + promotor???
+
:* Place three different constituive promotors with low, medium, and high activity in front of GVP-cluster
-
** PCR HmtA???
+
:::→ cutting the vector with promotor sequence resulted in a fragment of about 50bp and was difficult to locate on 1% agarose gel
-
** PCR GlpF???
+
:::→ 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)
-
** ArsR fusion???
+
:::→ {{todo}} 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
-
** Cloning strategy???
+
:* Isolate new plasmids of GVP and the three promotors to continu work
-
** Two variants of promotor+RBS (one with RBS from genome and one with RBS from GVP).???
+
:::→ grow new cultures of each from the glycerol stocks
-
** Create (and test) variants of GVP vector with/without Ori+resistance between the E and X sites???
+
:::→ {{todo}} determine concentration with nanodrop
 +
:* PCR HmtA???
 +
:* PCR GlpF???
 +
:* ArsR fusion???
 +
:* Cloning strategy (make a plan up to the final assembly)
 +
:::→ {{todo}} what are the target vectors going to be, and which genes are going to be grouped together
 +
:::→ {{todo}} what is still missing
 +
:::→ {{todo}} are there options to be investigated
 +
:* Two variants of promotor+RBS (one with RBS from genome and one with RBS from GVP).???
 +
:* {{todo}} Create (and test) variants of GVP vector with/without Ori+resistance between the E and X sites???
* [[:Category:Team:Groningen/Roles/Integrator|Integrator]]
* [[:Category:Team:Groningen/Roles/Integrator|Integrator]]
* [[:Category:Team:Groningen/Roles/Modeller|Modeller]]
* [[:Category:Team:Groningen/Roles/Modeller|Modeller]]

Revision as of 07:43, 14 July 2009

Igemhomelogo.png
Risk List Tools and Documentation Inception Elaboration Construction Transition
1 2 1 2 1 2 3 1 2
apr. 20 may 18 jun. 01 27 28 29 30 31 32 33 34 35 okt. 15

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:

  • TODO Our wonderful buoyant, metal filtering bacteria should be alive and kicking!
  • TODO (?) 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))
  • TODO The Iteration plan for the transition phase completed and reviewed.
  • TODO (?) 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))
  • TODO 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)
      1. Test insert length of gvp cluster (E-genR-X-RBS-PART-S-P in vector , Figure 1)
      2. (Needed?) Silence restrictionsites in gvp cluster by PCR (BamHI, XhoI, BglII --> used in other BBa standard assemblies)and remove repeat in gvpL.
      3. Add the terminator to the construct
      4. If needed add promoter (constitutive promoter --> ) to do initial phenotype testing.
    • Metal transporters (Jolanda, SJ, Nienke (?))
      1. 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).
      2. Test insert length.
      3. Silence restrictionsites in HtmA by PCR (2x PstI) and add BioBrick pre/suffix.
      4. Clone the terminator in to the construct.
    • Metal accumulation (Wilfred, Paul, Nienke (?))
      1. Transform E. coli with construct / synthetic gene.
      2. Test insert length.
      3. (If needed) Silence restrictionsites in gvp cluster by PCR.
      4. Add BioBrick pre/suffix by PCR.
      5. Clone the terminator in to the construct.
    • Vectors (Sven, Frans (Modelling aswell?)):
      1. Transform E. coli TOP10 with pSB3K3 and E. coli DB3.1 with pSB1AC3.
        1. pSB3K3 contains a p15A ORI and Kan resistance.
        2. pSB1AC3 contains Amp + Cam resistance markers, a ccdB death gene and a pMB1 ORI.
      2. Add different promoters to the vectors:
        1. Inducible are: pBAD-AraC in pSB2K3 and the Lac promoter in pSB1A2.
        2. Constitutive are: High expected expression yield, and Low expected expression yield, Lac promoter with mutated operator . For these promoter sequences are too small, oligo's have to be ordered + BBa pre/suffix !!without RBS!!
        3. Metal sentitive are possibly copA (order oligo's) and ??
          1. Clone in such a way that they are nicely -10 / -30 in front of the start codon.
      3. 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
    • TODO 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!)
    • DONE Reality check for buoyancy with arsenic.
    • Model accumulation of arsenic in E. coli.
      • TODO How well does ArsR(+fusion protein) bind to arsenic / its binding site.
      • TODO 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 :

  • 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)
TODO 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
  • Isolate new plasmids of GVP and the three promotors to continu work
→ grow new cultures of each from the glycerol stocks
TODO determine concentration with nanodrop
  • PCR HmtA???
  • PCR GlpF???
  • ArsR fusion???
  • Cloning strategy (make a plan up to the final assembly)
TODO what are the target vectors going to be, and which genes are going to be grouped together
TODO what is still missing
TODO are there options to be investigated
  • Two variants of promotor+RBS (one with RBS from genome and one with RBS from GVP).???
  • TODO Create (and test) variants of GVP vector with/without Ori+resistance between the E and X sites???
  • Integrator
  • Modeller
    • TODO 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!)
    • TODO Model import/export of arsenic in E. coli (use surface area per volume of cells + number of transporters per surface as measure?).
      • TODO Find diffusion speed (if nonzero).
      • TODO What (and how much) transporters in and out of the cell are there in normal E. coli.
      • TODO How much are we adding?
      • TODO "Reaction" rate(s) for in and out of the cell.
  • Project Manager
  • Public Relations Officer
  • Tester

Construction 2

Overall objective:

Tasks per role:

  • Analyst
  • Configuration Manager
  • Designer
  • Implementer and Tester
    • GVP (1 pers):
      1. Initial testing of phenotype, growth rates E. coli
      2. Clone gvp from BBa_J61035 to the high / low copy number vector
      3. (If the vectors do not already contain one) Clone gvp in vector with different promoters; high, moderate, low expression.
    • Metal transporters (2 pers):
      1. Initial testing of phenotype, growth rates E. coli.
      2. Clone HtmA to the high / low copy number vector.
      3. (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):
      1. Initial testing of phenotype, growth rates E. coli.
      2. Clone the gene to the high / low copy number vector.
      3. (If the vectors do not already contain one) Clone gvp in vector with different promoters; high, moderate, low expression (see GVP).
    • Vectors (1pers):
      1. Add different promoters to the vectors--> in such a way that they are nicely -10 / -30 in front of the start codon.
      2. 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 :

Week 31

July 27 - August 2 :

Week 32

August 3 - August 9 :

Construction 3

Overall objective:

Tasks per role:

  • Analyst
  • Configuration Manager
  • Designer
  • Implementer and Tester
    • GVP (1 pers):
      1. Test phenotype, growth rates E. coli again.
      2. Transform E. coli with both vectors
      3. 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)
      4. Make final buoyancy tests.
    • Metal transporters (2 pers):
      1. Test phenotype, growth rates E. coli again.
      2. Transform E. coli with both vectors.
      3. 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)
      4. Make final metal importing tests.
    • Metal accumulation (2 pers):
      1. Test phenotype, growth rates E. coli again.
      2. Transform E. coli with both vectors.
      3. 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)
      4. Make final metal accumulation tests.
  • Integrator
  • Modeller
  • Project Manager
  • Public Relations Officer

Week 33

August 10 - August 16 :

Week 34

August 17 - August 23 :

Week 35

August 24 - August 30 :