Arking:JCATutorialIntro1

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(What is Biobricks 2.0?)
Current revision (17:04, 7 December 2008) (view source)
(What is the BglBricks Standard?)
 
(10 intermediate revisions not shown.)
Line 16: Line 16:
The original version of Biobricks has a polylinker of EcoRI-XbaI-'''Part'''-SpeI-PstI.  SpeI and XbaI have "compatible cohesive ends".  What this means is that when digested, the DNA contains sticky ends of the same sequence, in this case CTAG overhangs.  XbaI-digested fragments can be ligated to SpeI-digested fragments.  When this occurs, both restriction sites are destroyed resulting in an ACTAGA scar sequence.  All methods of standard assembly somehow make these junctions between two parts.  However, there are multiply protocols being developed to implement the reaction.
The original version of Biobricks has a polylinker of EcoRI-XbaI-'''Part'''-SpeI-PstI.  SpeI and XbaI have "compatible cohesive ends".  What this means is that when digested, the DNA contains sticky ends of the same sequence, in this case CTAG overhangs.  XbaI-digested fragments can be ligated to SpeI-digested fragments.  When this occurs, both restriction sites are destroyed resulting in an ACTAGA scar sequence.  All methods of standard assembly somehow make these junctions between two parts.  However, there are multiply protocols being developed to implement the reaction.
-
===What is the BBb Standard?===
+
http://partsregistry.org/wiki/images/2/22/StdAsssmall.png<br>
-
BBa is much like the original (BBa), but the polylinker is EcoRI-BglII-'''Part'''-BamHI-XhoI.  The main advantage of this standard is that the scar sequence is GGATCT.  When translated in the zero frame, this becomes Gly-Ser, which is a good linker sequence for peptide fusions.  BBb can therefore be used for translational fusions.  Additionally, the enzymes in BBb digest more efficiently than the BBa enzymes and are not blocked by dam methylation.
+
Schematic of Biobrick standard assembly.  Any number of parts in any order are joined in a pairwise manner using ligation of compatible cohesive ends.  Image from partsregistry.org.
 +
 
 +
===What is the BglBricks Standard?===
 +
BglBricks (formally referred to as BBb) is much like the original XbaI/SpeI standard, but the polylinker is EcoRI-BglII-'''Part'''-BamHI-XhoI.  The main advantage of this standard is that the scar sequence is GGATCT.  When translated in the zero frame, this becomes Gly-Ser, which is a good linker sequence for peptide fusions.  BglBricks can therefore be used for translational fusions.  Additionally, the enzymes used in BglBricks are highly efficient and are not blocked by dam methylation.  The homepage describing this standard is [[BBb | here]]. 
 +
 
 +
For the purposes of this tutorial, what you need to know about BglBricks is that you want your plasmids to look like this:
 +
 
 +
GAATTCatgAGATCT-part-GGATCCtaaCTCGAG
 +
 
 +
So, if you are going to clone a new part into the EcoRI (GAATTC) and BamHI (GGATCC) sites of a vector, you'll want to include the BglII (AGATCT) site and the "ATG" spacer in your oligos so that it is Biobricked properly.
 +
 
 +
In this tutorial, we'll look at examples of the original Biobrick standard as well as BglBrick examples.  The different standards are all pretty much the same in terms of how you go about designing and executing the cloning experiment.  So, if you understand one, you understand them all.  When performing the quizes, pay special attention to which standard is requested and make sure you design the part such that it obeys the standard.
So, to do Biobricks, the general routine is to make plasmids containing Basic Parts, and then use methods of standard assembly to construct Composite Parts.  The remainder of this tutorial will address the question of how you do these things.
So, to do Biobricks, the general routine is to make plasmids containing Basic Parts, and then use methods of standard assembly to construct Composite Parts.  The remainder of this tutorial will address the question of how you do these things.
{{Template:JCA_Arkin_tutorialfooter}}
{{Template:JCA_Arkin_tutorialfooter}}

Current revision

Introduction to Biobricks 2.0

What is Biobricks?

Biobricks is a programming language for Synthetic Biology (SB). SB means ground-up genetic or cellular Engineering. We add DNA sequences into well-characterized model organisms to understand biological processes or create useful organisms. So, we're working principally with E. coli, yeast, human cells, or model plant cells rather than exotic natural isolates. This restriction on organism allows us develop a shared engineering knowledge. If you and I both do our experiments in the same strain of E. coli, we can understand each other’s results, and perhaps combine them into more advanced systems. The modifications we make to these organisms could be the addition of DNAs or the removal or replacement of DNAs present in the genome. These latter types of modifications are termed "chassis" modifications. There is no method yet for standardization of chassis modification. Biobricks is a standard language for building and describing the DNAs for the former type of modification.

Biobricks begins with defining specific sequences termed basic parts. These basic parts are flanked by a standard restriction enzyme-based polylinker in an appropriate plasmid. So, a Biobrick basic part is a DNA sequence. It's a conceptual beast, not a physical thing. A physical plasmid containing the Biobrick sequence is just a plasmid. In this way, a basic part is much like a word in normal language. It's a string of letters (A,T,C, or G) whereas English words have 26 letter choices. Like English words, Biobricks are assembled into larger sentences (called devices) in a linear order. Basic parts alone rarely confer any activity to a cell just as words alone confer little meaning. Biobrick basic parts only confer activity in a cell when other appropriate parts flank the part. Just as English follows a hierarchical organization of letters > words > sentences > paragraphs > books > libraries, Biobricks follow a hierarchy of base pairs > basic parts > composite parts > systems > ecosystems. The language and relevant descriptions of this hierarchy are still being developed in the SB field, but for now, all DNAs are either basic parts or composite parts.

You can learn more basic information about Biobricks at The registry

Why use Biobricks?

The reason for doing Biobricks is 3-fold. First of all, there is a standard assembly method associated with Biobricks. We'll get into this later, but the general idea is that if everyone uses similar methods of assembly, we open up the possibility that researchers or companies can develop faster, cheaper ways of assembling composite parts. Secondly, this standardization results in a set of parts that become a shared resource readily usable by the entire biology community. The third reason is that it rigorously defines the composition of a design which will enable quantitative standardization and descriptions of robustness. Consider the following figure:

This is a typical example of the way sequences of DNA are described throughout the biological disciplines, and there's nothing necessarily wrong with it. Consider, though, that someone handed you this map and told you to make something that conferred whatever activity this thing confers. You see that it has an SV40 origin. So, maybe you'd pubmed search SV40 and find some annotation of what this thing should be. Often in doing this, you'll find maybe 10 different sequences all annotated as the SV40 origin. These labels of function therefore rarely define a unique sequence. Also, consider that you were going to clone this origin into a plasmid. Where would you "cut" the sequence? How many base pairs upstream and downstream of the annotated sequence would you include? In this case, it might not matter, but when describing features such as promoter and terminator elements this is entirely hazy. Biobricks resolves this issue. DNA elements are no longer defined with abstract terms such as SV40 origin, they become a specific Biobrick sequence with an associated number such as J23997. There may be 100 different SV40 origin-like Biobrick parts, but when you construct an actual plasmid, you pick a specific Biobrick and the composite part you construct can be rigorously defined by the linear sequence of Biobrick parts from which it was made. This allows us to decipher the basic elements that robustly confer their function in a variety of contexts. Consider two different (hypothetical) SV40 origin parts J23997 and I13597. Let's say that J23997 "worked" in 23 out of 50 cases. Say I13597 worked in 43 of our 50 cases. Which one would you then choose if you needed to make a composite part with an SV40 origin? So, basically, this allows us to share parts and also encapsulate information about the use of parts.

Standard Assembly

The original version of Biobricks has a polylinker of EcoRI-XbaI-Part-SpeI-PstI. SpeI and XbaI have "compatible cohesive ends". What this means is that when digested, the DNA contains sticky ends of the same sequence, in this case CTAG overhangs. XbaI-digested fragments can be ligated to SpeI-digested fragments. When this occurs, both restriction sites are destroyed resulting in an ACTAGA scar sequence. All methods of standard assembly somehow make these junctions between two parts. However, there are multiply protocols being developed to implement the reaction.

StdAsssmall.png
Schematic of Biobrick standard assembly. Any number of parts in any order are joined in a pairwise manner using ligation of compatible cohesive ends. Image from partsregistry.org.

What is the BglBricks Standard?

BglBricks (formally referred to as BBb) is much like the original XbaI/SpeI standard, but the polylinker is EcoRI-BglII-Part-BamHI-XhoI. The main advantage of this standard is that the scar sequence is GGATCT. When translated in the zero frame, this becomes Gly-Ser, which is a good linker sequence for peptide fusions. BglBricks can therefore be used for translational fusions. Additionally, the enzymes used in BglBricks are highly efficient and are not blocked by dam methylation. The homepage describing this standard is here.

For the purposes of this tutorial, what you need to know about BglBricks is that you want your plasmids to look like this:

GAATTCatgAGATCT-part-GGATCCtaaCTCGAG

So, if you are going to clone a new part into the EcoRI (GAATTC) and BamHI (GGATCC) sites of a vector, you'll want to include the BglII (AGATCT) site and the "ATG" spacer in your oligos so that it is Biobricked properly.

In this tutorial, we'll look at examples of the original Biobrick standard as well as BglBrick examples. The different standards are all pretty much the same in terms of how you go about designing and executing the cloning experiment. So, if you understand one, you understand them all. When performing the quizes, pay special attention to which standard is requested and make sure you design the part such that it obeys the standard.

So, to do Biobricks, the general routine is to make plasmids containing Basic Parts, and then use methods of standard assembly to construct Composite Parts. The remainder of this tutorial will address the question of how you do these things.


If you have any comments or want to report a potential error in the tutorial, please email me (Chris Anderson) at JCAnderson2167-at-gmail.com

Personal tools