User:Michael Verhoeven/Notebook/iGEM 2009 M. Verhoeven Parts

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(Project Description/Abstract)
(Notes)
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==Notes==
==Notes==
-
'''Transformation of ''E. coli TOP10'' with BBa_J23109, BBa_J23100 and BBa_J23106'''
 
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* BBa_J23109 iGEM plate 2, 2G
+
We are working on part [http://partsregistry.org/wiki/index.php?title=Part:BBa_I750016 BBa_I750016], a biobrick submitted by [http://parts.mit.edu/igem07/index.php/Melbourne/Lab_GV_Notebook Melbourne iGEM 2007 team]. The part consists of: gvpB, gvpR, gvpN, gvpF, gvpG, gvpL, gvpS, gvpK, gvpJ, gvpT and gvpU in vector [http://partsregistry.org/wiki/index.php?title=Part:BBa_J61035 BBa_J61035]. The gvpL part contains a 40bp repeat, which is repeated 10 times.
-
* BBa_J23100 iGEM plate 1, 18C
+
-
* BBa_J23106 iGEM plate 1, 18O
+
-
All dry plasmid DNA in the wells was dissolved in 15ul sterile diH2O, and transfered to 500ul cups.
+
'''Future directions of Melbourne'''
-
* Four cups with 50ul competent ''E.coli TOP10'' cells were thawed on ice, and 2ul of dissolved plasmid was added.
+
* smaller functional biobrick
-
* After 30 min. on ice, the cups were put in a heat block at 37C for 5 min. and followed by 5 min. on ice.
+
:→ design primers for ligation strategy to knock out individual genes
-
* To the cells, 800ul TY medium was added and incubated at 37C for 1 hour.
+
* literature suggests gvpN might be a negative regulator of expression
-
* 100ul was plated on TY-Agar-Amp plates and incubated o.n. at 37C.
+
:→ knock-out might produce truly bouyant bacteria
-
* The remainer was centrifuged for 1 min. at full speed and the cells were resuspended in 100ul TY-medium, and plated on TY-Agar-Amp plated. The plates were stored at 37C o.n.
+
* phenotype not affected by gvpL insert of repeat
 +
:→ why?
 +
* gvpA and gvpC are to be included in mimimal set of 8 genes according to literature, but they are not present in biobrick
 +
:→ does gvpB have a similar function, and can it be substituted by gvpA or gvpC?
-
'''Inoculation of TY-medium with Glycerol Stocks'''
+
'''Own plan of action'''
-
* GVP cluster vector BBa_J61035 (2x) in ''E.coli TOP10''
+
* test insert length of gvp cluster in biobrick
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* Terminator vector ? (2x) in ''E.coli TOP10''
+
:→ E-genR-X-RBS-part-S-P in vector [http://partsregistry.org/wiki/index.php?title=Part:BBa_J61035 BBa_J61035]
 +
* check cluster for restriction sites
 +
:→ EcoRI, XbaI, SpeI and PstI are not allowed and not present in part
 +
:→ other sites might become a problem in future assembly protocols, and need to be silenced
 +
* add additional parts
 +
:→ promotors (constitutive, metal sensitive....)
 +
:→ RBS (substitute exsisting one)
 +
:→ add gvpA and/or gvpC, knock-out gvpN
 +
* test initial phenotype with different constitutive promotors
-
* 4.5 ml TY-medium with Amp. was inoculated with a colony grown on TY-Agar-Amp. plates o.n.
 
-
* The tubes were put in a waterbath at 37C (150 rpm) and grown o.n.
 

Revision as of 08:20, 9 July 2009

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Project Description/Abstract

  • Bouyant Bacteria Project (BBP)
  • GVP is a cluster of 14 genes involved in gas vesicle production in B. megaterium and has been succesfully expressed in E.coli. Not all genes are of critical importance and at least 8 genes are needed. Our aim is to improve the bouyant capacity of the vesicles in E.coli.

Notes

We are working on part BBa_I750016, a biobrick submitted by Melbourne iGEM 2007 team. The part consists of: gvpB, gvpR, gvpN, gvpF, gvpG, gvpL, gvpS, gvpK, gvpJ, gvpT and gvpU in vector BBa_J61035. The gvpL part contains a 40bp repeat, which is repeated 10 times.

Future directions of Melbourne

  • smaller functional biobrick
→ design primers for ligation strategy to knock out individual genes
  • literature suggests gvpN might be a negative regulator of expression
→ knock-out might produce truly bouyant bacteria
  • phenotype not affected by gvpL insert of repeat
→ why?
  • gvpA and gvpC are to be included in mimimal set of 8 genes according to literature, but they are not present in biobrick
→ does gvpB have a similar function, and can it be substituted by gvpA or gvpC?

Own plan of action

  • test insert length of gvp cluster in biobrick
→ E-genR-X-RBS-part-S-P in vector BBa_J61035
  • check cluster for restriction sites
→ EcoRI, XbaI, SpeI and PstI are not allowed and not present in part
→ other sites might become a problem in future assembly protocols, and need to be silenced
  • add additional parts
→ promotors (constitutive, metal sensitive....)
→ RBS (substitute exsisting one)
→ add gvpA and/or gvpC, knock-out gvpN
  • test initial phenotype with different constitutive promotors


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