CIDARWiki - Recent changes [en]

File:Promoters1.png

thaddock — Wed, 16 May 2012 19:10:56 GMT

New page

Projects/Design Drivers/Parts

thaddock — Wed, 16 May 2012 19:10:47 GMT

Promoters

← Older revision		Revision as of 19:10, 16 May 2012
(4 intermediate revisions not shown)
Line 1:		Line 1:
	__TOC__		__TOC__
		+
		+	==Introduction to Characterizing DNA Parts==
		+
		+

	==Promoters==		==Promoters==

-	[[Image:~~promoters~~.png \| ~~500px~~\| center]]	+
		+	[[Image:promoters1.png \| 650px\| center]]

	==Ribosomal Binding Sites==		==Ribosomal Binding Sites==

-	[[Image:rbs.png \| ~~500px~~\| center]]	+	[[Image:rbs.png \| 650px\| center]]

	==Order Effect==		==Order Effect==

-	[[Image:order.png \| ~~500px~~\| center]]	+	[[Image:order.png \| 800px\| center]]

File:Order.png

thaddock — Wed, 16 May 2012 17:47:22 GMT

uploaded "[[File:Order.png]]"

New page

Projects/Design Drivers/Parts

thaddock — Wed, 16 May 2012 17:46:25 GMT

New page

__TOC__

==Promoters==

[[Image:promoters.png | 500px| center]]

==Ribosomal Binding Sites==

[[Image:rbs.png | 500px| center]]

==Order Effect==

[[Image:order.png | 500px| center]]

Projects

thaddock — Wed, 16 May 2012 14:49:29 GMT

Design Drivers

← Older revision		Revision as of 14:49, 16 May 2012
(3 intermediate revisions not shown)
Line 74:		Line 74:
	*[[Projects/Design Drivers/Invertase\|Invertase]] based computation.		*[[Projects/Design Drivers/Invertase\|Invertase]] based computation.
	*[[Projects/Design Drivers/CombLogic\|Combinational Logic]] based computation.		*[[Projects/Design Drivers/CombLogic\|Combinational Logic]] based computation.
-		+	*[[Projects/Design Drivers/Parts\|Parts Characterization]] is where the function of various promoters, ribosomal binding sites, and genes is determined using FACS.
	\|}		\|}

MoClo

thaddock — Wed, 16 May 2012 14:39:53 GMT

Making Level 2 Modules

← Older revision		Revision as of 14:39, 16 May 2012
(One intermediate revision not shown)
Line 35:		Line 35:
	==Making Level 2 Modules==		==Making Level 2 Modules==

-	[[Image:MoClo_2.jpg \| ~~800px~~\| center]]	+	[[Image:MoClo_2.jpg \| 1200px\| center]]

	==References==		==References==
	[http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0016765 A Modular Cloning System for Standardized Assembly of Multigene Constructs, PloS One 2011]		[http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0016765 A Modular Cloning System for Standardized Assembly of Multigene Constructs, PloS One 2011]

File:MoClo 2.jpg

thaddock — Wed, 16 May 2012 14:39:21 GMT

uploaded "[[File:MoClo 2.jpg]]"

New page

MoClo

thaddock — Wed, 16 May 2012 14:38:39 GMT

← Older revision		Revision as of 14:38, 16 May 2012
(5 intermediate revisions not shown)
Line 20:		Line 20:


-	[[Image:MoClo_DV.png \| 800px\| center\|]]	+	[[Image:MoClo_DV.png \| 800px\| center]]


Line 30:		Line 30:
	==Making Level 0 and Level 1 Modules==		==Making Level 0 and Level 1 Modules==

-	[[Image:MoClo_mod.jpg \| 800px\| center]]	+	[[Image:MoClo_mod.jpg \| 1200px\| center]]
		+
		+
		+	==Making Level 2 Modules==
		+
		+	[[Image:MoClo_2.jpg \| 800px\| center]]

	==References==		==References==
	[http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0016765 A Modular Cloning System for Standardized Assembly of Multigene Constructs, PloS One 2011]		[http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0016765 A Modular Cloning System for Standardized Assembly of Multigene Constructs, PloS One 2011]

File:MoClo mod.jpg

thaddock — Wed, 16 May 2012 14:34:34 GMT

uploaded "[[File:MoClo mod.jpg]]"

New page

MoClo

thaddock — Wed, 16 May 2012 14:34:22 GMT

Making Level 0 and Level 1 Modules

← Older revision		Revision as of 14:34, 16 May 2012
(2 intermediate revisions not shown)
Line 28:		Line 28:


-	==Making Level 0 to Level 1 Modules==	+	==Making Level 0 and Level 1 Modules==
		+
		+	[[Image:MoClo_mod.jpg \| 800px\| center]]

-	~~[[Image:MoClo_01.png \| 800px\| center\|]]~~
	==References==		==References==
	[http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0016765 A Modular Cloning System for Standardized Assembly of Multigene Constructs, PloS One 2011]		[http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0016765 A Modular Cloning System for Standardized Assembly of Multigene Constructs, PloS One 2011]

File:MoClo 01.png

thaddock — Wed, 16 May 2012 14:14:08 GMT

uploaded "[[File:MoClo 01.png]]"

New page

MoClo

thaddock — Wed, 16 May 2012 14:13:55 GMT

← Older revision		Revision as of 14:13, 16 May 2012
(6 intermediate revisions not shown)
Line 3:		Line 3:
	==Modular Cloning or MoClo==		==Modular Cloning or MoClo==

-	MoClo is a relatively new assembly method introduced in 2011 by Ernst Weber et al., whereby using Type IIs restriction sites allows the user to ligate up to six DNA parts together in a one-pot reaction. Type IIs restriction enzymes cleave outside of their recognition site to one side, thus allowing for removal of those restriction sites when used properly. This helps eliminate excess base pairs, or scars, from forming between DNA Parts. However, in order to ligate together properly, MoClo utilizes 4-bp fusion sites, which ~~end up remaining~~ behind and thus generate 4-bp scars between DNA parts in the final DNA sequence following ligation of two or more parts.	+	MoClo is a relatively new assembly method introduced in 2011 by Ernst Weber et al., whereby using Type IIs restriction sites allows the user to ligate up to six DNA parts together in a one-pot reaction. Type IIs restriction enzymes cleave outside of their recognition site to one side, thus allowing for removal of those restriction sites when used properly. This helps eliminate excess base pairs, or scars, from forming between DNA Parts. However, in order to ligate together properly, MoClo utilizes a set of 4-bp fusion sites, which remain behind after ligation and thus generate 4-bp scars between DNA parts in the final DNA sequence following ligation of two or more parts.
		+
		+	The MoClo system has three levels of assembly.
		+
		+	* Level 0: Basic DNA Parts (ex: promoter, gene, etc.) are PCR amplified and then cloned into MoClo destination vectors to form Level 0 Modules. The DNA parts within these Level 0 Modules are flanked by BsaI sites and two different 4pb-fusion sites.
		+
		+	* Level 1: Up to six Level 0 parts are ligated together to form Level 1 Modules. In our lab, Level 1 Modules most often result in complete transcriptional units (ex: promoter-RBS-gene-terminator). Level 1 Modules are flanked by BpiI sites and two different 4pb-fusion sites.
		+
		+	* Level 2: Up to six Level 1 Modules are ligated together to form Level 2 Modules. More complex circuits, such as an inverter or NOR gate, can be built using Level 2 Modules. Like Level 0 Modules, Level 2 Modules are flanked by BsaI sites and two different 4pb-fusion sites.
		+
		+	Below we discuss how MoClo is used in our lab to create genetic circuits.

	==Making MoClo Plasmids==		==Making MoClo Plasmids==
Line 10:		Line 20:


-	[[Image:MoClo_DV.png \| center\|]]	+	[[Image:MoClo_DV.png \| 800px\| center\|]]
		+
		+
		+	The end result shown here is a Level 0 DV with the proper MoClo Type IIs restriction sites (BsaI and BpiI) flanking the LacZ region, with the BioBrick restriction sites (EcoRI, XbaI, SpeI and PstI) flanking the Type IIs restriction sites. It should be noted that the only BioBrick restriction site not found in the LacZ region was SpeI. As a result, we had to clone the LacZ region into the SpeI site of the BioBrick plasmids and then subsequently sequence our clones to select ones that have the LacZ region in the proper orientation.
		+
		+	Level 1 DVs are made the same way except the order of BsaI and BpiI is flipped and the backbone has ampicillin resistance. Level 2 DVs resemble Level 0 DVs except they have chloramphenicol resistance.
		+
		+
		+	==Making Level 0 to Level 1 Modules==

		+	[[Image:MoClo_01.png \| 800px\| center\|]]
	==References==		==References==
	[http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0016765 A Modular Cloning System for Standardized Assembly of Multigene Constructs, PloS One 2011]		[http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0016765 A Modular Cloning System for Standardized Assembly of Multigene Constructs, PloS One 2011]

File:MoClo DV.png

thaddock — Wed, 16 May 2012 13:52:51 GMT

uploaded "[[File:MoClo DV.png]]"

New page

MoClo

thaddock — Wed, 16 May 2012 13:52:36 GMT

Making MoClo Plasmids

← Older revision		Revision as of 13:52, 16 May 2012
(5 intermediate revisions not shown)
Line 1:		Line 1:
-	[[~~File~~:~~MoClo_paper~~.~~pdf~~]]	+	__TOC__
		+
		+	==Modular Cloning or MoClo==
		+
		+	MoClo is a relatively new assembly method introduced in 2011 by Ernst Weber et al., whereby using Type IIs restriction sites allows the user to ligate up to six DNA parts together in a one-pot reaction. Type IIs restriction enzymes cleave outside of their recognition site to one side, thus allowing for removal of those restriction sites when used properly. This helps eliminate excess base pairs, or scars, from forming between DNA Parts. However, in order to ligate together properly, MoClo utilizes 4-bp fusion sites, which end up remaining behind and thus generate 4-bp scars between DNA parts in the final DNA sequence following ligation of two or more parts.
		+
		+	==Making MoClo Plasmids==
		+
		+	In order to utilize MoClo in the lab, first a set of plasmids, or destination vectors (DV), must be generated. We used BioBrick plasmids that we already had as the backbone for the MoClo DVs. We then PCR amplified a LacZ alpha coding region found in pUC19 that will allow for blue/white screening and cloned the PCR product into the BioBrick backbones, thus generating our own MoClo destination vectors.
		+
		+
		+	[[Image:MoClo_DV.png \| center\|]]
		+
		+	==References==
		+	[http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0016765 A Modular Cloning System for Standardized Assembly of Multigene Constructs, PloS One 2011]

File:MoClo paper.pdf

thaddock — Wed, 16 May 2012 13:26:51 GMT

uploaded "[[File:MoClo paper.pdf]]"

New page

MoClo

thaddock — Wed, 16 May 2012 13:26:02 GMT

Created page with 'File:MoClo_paper.pdf'

New page

[[File:MoClo_paper.pdf]]

Projects

thaddock — Wed, 16 May 2012 13:24:16 GMT

Assembly

← Older revision		Revision as of 13:24, 16 May 2012
Line 55:		Line 55:
	**[[Projects/Assembly/Puppeteer\|Puppeteer]] is a language for specifying biological protocols and a software stack for executing them on a liquid handling robot.		**[[Projects/Assembly/Puppeteer\|Puppeteer]] is a language for specifying biological protocols and a software stack for executing them on a liquid handling robot.
	**[[Projects/Assembly/PuppeteerShow\|Puppet Show]] is a Clotho App.		**[[Projects/Assembly/PuppeteerShow\|Puppet Show]] is a Clotho App.
		+
		+	* Assembly Methods
		+	**[[MoClo\|MoClo]] is an assembly method that allows the user to ligate up to six DNA Parts together in one reaction
	\|}		\|}

Main Page

dougd — Tue, 15 May 2012 16:47:13 GMT

← Older revision		Revision as of 16:47, 15 May 2012
(3 intermediate revisions not shown)
Line 33:		Line 33:

	<!--<br clear="all">-->		<!--<br clear="all">-->
-	*5/8/12 - [http://www.defense.gov/releases/release.aspx?releaseid=15254 Office of Naval Research Defense University Research Instrumentation Program (DURIP)] awards the proposal ''Flow Cytometry Machinery to Enable Characterization Driven Synthetic Biology Software Design Flow'' $419,220 for equipment to create a comprehensive bio-design automation framework.	+	*5/9/12 - Boston University's Dean's Catalyst Award granted for ''Microfluidic Biocomputing: Large-Scale Biofluidic Circuits for Programmable Logic Devices'' along with [http://www.bu.edu/khalillab/ Prof. Mo Khalil].
		+
		+
		+	*5/8/12 - [http://www.defense.gov/releases/release.aspx?releaseid=15254 Office of Naval Research Defense University Research Instrumentation Program (DURIP)] awards the proposal ''Flow Cytometry Machinery to Enable Characterization Driven Synthetic Biology Software Design Flow'' $419,220 for equipment to create a comprehensive bio-design automation framework. For more click [http://www.bu.edu/ece/2012/05/15/onr-awards-densmore/ here].