Data

iGEM UT-Tokyo

Data

Method

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Modeling

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Parts submitted

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Lab Note

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How our system works

Fig.1 SMART E. coli

(A) Substrate-induced cell assembling system
1. Substrate(IPTG) induces expression from the Lac promoter.
2. AspA is expressed.
3. AspA catalyses the production of L-Asp from fumaric acid and ammonium.
(B) Substrate-induced cell arrest system
cheZ-/- strain is used.
4. The cI promoter is active when the cI inhibitor is absent.
5. CheZ is expressed and rescues motility of CheZ- strain.
6. When cI inhibitor is expressed it inhibits the activity of cI promotor. This results in the repression of CheZ expression, which leads to loss of motility.

Fig.2 Dual luciferase assay

1. A constitutive promoter of known strength is placed upstream of renilla. and a promoter of interest is placed upstream of firefly.
2. By adding the oxidizing substrate D-luciferin, firefly luminescence is measured.
3. Another oxidizing substrate, coelenterazine, is added to measure renilla luminescence as a reference to compute the relative strength of the promoter upstream of the firefly luciferase.

Data for our favourite new parts

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K518007 BBa_K518007] cheZ expression cassette (no promoter)
This construct rescues the mobility of cheZ-/- cells.

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K518010 BBa_K518010] sulA promoter
This is a promoter of the sulA gene which is responsible for stress-induced arrest of cell division. We successfully demonstrated a significant alteration of expression of a gene downstream of this promoter after UV irradiation

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K518013 BBa_K518013]　sulA promoter evaluation device
sulAp is known to respond to various types of DNA-injuring stress. This construct is a sulAp evaluation device to make it easy to compare the expression of sulAp in various "stressful" conditions. Employing our dual luciferase assay kit, both quantitative measurements and a comparison of Relative Promoter Unit (RPU) can be achieved.

Data for pre-existing parts

[http://partsregistry.org/Part:BBa_I712019:Experience BBa_I712019] Firefly luciferase - luciferase from Photinus pyralis (Ljubljana, 2007)
This is firefly luciferase which produces luminescence by oxidation of D-luciferin.
We utilized this part to devise a Firefly-Renilla Dual Luciferase Assay Kit.

[http://partsregistry.org/Part:BBa_J52008:Experience BBa_J52008] luciferase: luciferin 2-monooxygenase from Renilla reniformis (Slovenia, 2006)
This is Renilla luciferase which emits luminescence when coelenterazine is added.
We utilized this part to devise a Firefly-Renilla Dual Luciferase Assay Kit.

[http://partsregistry.org/Part:BBa_R0011:Experience BBa_R0011] Promoter (lacI regulated, lambda pL hybrid) (Neelaksh Varshney et al., 2003)
We evaluated the relative expression levels of this promoter with various IPTG concentrations.

We’ve also characterized the following parts

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K518000 BBa_K518000] RBS + firefly luciferase + d.terminator
Firefly luciferase emits luminescence by the oxidation of D-luciferin. This construct can be used as a measuring tool when combined with other cis-elements, because of its high quantitative performance.

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K518001 BBa_K518001]　RBS + renilla luciferase + d.terminator
Renilla luciferase emits luminescence by the oxidation of coelenterazine. This construct can be used as a measuring tool when combined with other cis-elements, because of its high quantitative performance.

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K518002 BBa_K518002] Firefly-renilla dual luciferase assay kit
This construct enables Luciferase assay which is one of the most popular reporter assay system for quantitatively measuring the strength of promoters and other cis-elements. The wide-range and quantitative detection are the prominent features of this assay. A promoter or other cis-elements to be analysed can be ligated upstream of this part.

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K518003 BBa_K518003] LexA - a repressor for LexA-regulated SOS promoters
LexA protein represses most of SOS genes which are activated in case of DNA damage.

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K518004 BBa_K518004] IPTG-inducible L-Asp producing device
This construct enables production of aspartate in the presence of enough substrate (fumaric acid and NH4+).
IPTG can be used to induce aspartate production.

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K518005 BBa_K518005] cheZ
CheZ is responsible for the dephosphorylation of the flagellum-regulating protein CheY Non-phosphorylated CheY results in E. coli swimming straight.

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K518006 BBa_K518006] IPTG-inducible CheZ expression device
This construct rescues cheZ-/- cell motility in the presence of IPTG.

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K518008 BBa_K518008] IPTG-inducible CheZ repression device
This construct usually allows strong cheZ expression, while in the presence of IPTG, the transcription of cheZ is repressed by the lambda cI repressor.

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K518009 BBa_K518009] AspA expression cassette (no promoter)
This construct enables the production of aspartate in the presence of enough substrate (fumaric acid and NH4+).

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K518011 BBa_K518011] recN promoter
This is a promoter of the recN gene which is involved in repair of double stranded breaks in the chromosome.

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K518012 BBa_K518012] RBS + RFP + d.Ter
This part is an easy promoter assessment tool. The RFP reporter aids you to find out whether a promoter of your interest works.