Team:UT-Tokyo/Data

From 2011.igem.org

(Difference between revisions)
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_K518008 BBa_K518008]  '''IPTG-inducible CheZ repression device'''<br>
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K518008 BBa_K518008]  '''IPTG-inducible CheZ repression device'''<br>
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This construct usually allows strong cheZ expression, while in the presence of IPTG, the transcription of cheZ is repressed by the lambda cI repressor.
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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_K518012 BBa_K518012]  '''RBS + RFP + d.Ter'''<br>
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K518012 BBa_K518012]  '''RBS + RFP + d.Ter'''<br>

Revision as of 01:12, 6 October 2011

How our system works

SMART E.coli

Figure 1. SMART E.coli
Figure 1. SMART E.coli

(A) Substrate-induced Cell Assembling System
1. Substrate (this time, an IPTG) induces expression from a substrate-induced promoter (BBa_R0011).
2. aspA (BBa_C0083) is expressed.
3. AspA catalyses the production of L-aspartate from fumarate and ammonium ion.
(B) Substrate-induced Cell Arrest System
cheZ-/- strain is cloned for the works below.
4. The cI-repressed promoter (BBa_R0051) is active when the cI repressor is absent.
5. CheZ is expressed and rescues the motility of cheZ-/- strain.
6. When cI repressor (BBa_C0051) is induced, it inhibits the activity of cI promotor. This results in the repression of CheZ expression, which leads to the loss of motility.


Dual luciferase assay

Figure 2. Dual luciferase assay
Figure 2. Dual luciferase assay

1. A constitutive promoter of known strength (this time, BBa_J23118) is placed upstream of renilla luciferase expression cassette (BBa_K518001), and a promoter of interest is placed upstream of firefly luciferase expression cassette (BBa_K518000).
2. After an instillation of first substrate D-luciferin, firefly luminescence is measured.
3. After another substrate, coelenterazine, is added, renilla luminescence is measured as a reference to compute the ratio of the expression level of the target promoter to that of the internal control promoter (BBa_J23118).

Data for our favourite new parts

BBa_K518007 cheZ expression cassette (no promoter)
This construct rescues the mobility of cheZ-/- cells.

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. This promoter can be utilized in our system.

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

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.

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.

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

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.

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.

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.

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.

BBa_K518005 cheZ
CheZ is responsible for the dephosphorylation of the flagellum-regulating protein CheY.
Non-phosphorylated CheY results in E. coli swimming straight.

BBa_K518006 IPTG-inducible CheZ expression device
This construct rescues cheZ-/- cell motility in the presence of IPTG.

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.

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.