Difference between revisions of "CH391L/S14/BioBricks"
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− | + | = What are BioBricks, and what is their purpose? = | |
A BioBrick is a sequence of DNA with a predefined structure and function. The information is held in a plasmid, or a circular piece of DNA that can be inserted, and replicated into bacteria. | A BioBrick is a sequence of DNA with a predefined structure and function. The information is held in a plasmid, or a circular piece of DNA that can be inserted, and replicated into bacteria. | ||
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− | + | = The creation of a standard sequence interface for all BioBricks: = | |
The original BioBrick assembly standard created by Tom Knight: | The original BioBrick assembly standard created by Tom Knight: | ||
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− | + | = Restriction Endonucleases = | |
Restriction enzymes, or restriction endonucleases are enzymes that cleave DNA at restriction sites. A restriction site is a sequence of base pairs (generally 4-8 base pairs in length) that is recognized and spliced by a restriction enzyme. To cut DNA, restriction enzymes make two incisions: once through each side of the sugar-phosphate backbone. There are 5 kinds of restriction enzymes. | Restriction enzymes, or restriction endonucleases are enzymes that cleave DNA at restriction sites. A restriction site is a sequence of base pairs (generally 4-8 base pairs in length) that is recognized and spliced by a restriction enzyme. To cut DNA, restriction enzymes make two incisions: once through each side of the sugar-phosphate backbone. There are 5 kinds of restriction enzymes. | ||
− | + | ==Types of Restriction Endonucleases== | |
Type I: These were the first type of enzymes to be discovered, but have very little use in synthetic biology because they randomly cleave the DNA varying distances from the restriction site. The recognition site is composed of two different, asymmetrical portions: one containing 3-4 nucleotides, and another containing 4-5 nucleotides, and are separated by a non-specific spacer that is composed of 6-8 nucleotides. They are a multifunctional protein with both methylase and restriction activity. | Type I: These were the first type of enzymes to be discovered, but have very little use in synthetic biology because they randomly cleave the DNA varying distances from the restriction site. The recognition site is composed of two different, asymmetrical portions: one containing 3-4 nucleotides, and another containing 4-5 nucleotides, and are separated by a non-specific spacer that is composed of 6-8 nucleotides. They are a multifunctional protein with both methylase and restriction activity. | ||
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Type III: | Type III: | ||
− | + | ==Considerations in the selection of Restriction Enzymes== | |
'''List of Restriction Enzymes used with BioBricks''' | '''List of Restriction Enzymes used with BioBricks''' | ||
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− | + | = BioBrick composition techniques= | |
There are two methods of composing a biobrick component: Prefixing one component to another, or postfixing one component with another. Both techniques result in a new, compound component, which can then be used as either an insert or vector in other reactions. | There are two methods of composing a biobrick component: Prefixing one component to another, or postfixing one component with another. Both techniques result in a new, compound component, which can then be used as either an insert or vector in other reactions. | ||
− | + | ==Prefixing a recipient vector with a donor component == | |
In order to prefix a recipient component, a front vector is created. To do so, the component is cut with EcoRI and XbaI enzymes: | In order to prefix a recipient component, a front vector is created. To do so, the component is cut with EcoRI and XbaI enzymes: | ||
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The EcoRI and XbaI sites are recreated, and a mixed, uncuttable SpeI/XbaI site is created. The other restriction regions SpeI and PstI remain untouched. | The EcoRI and XbaI sites are recreated, and a mixed, uncuttable SpeI/XbaI site is created. The other restriction regions SpeI and PstI remain untouched. | ||
− | + | ==Postfixing a recipient vector with a donor component== | |
A back vector is created with SpeI and PstI to insert a donor fragment after a component on a vector. This vector is created: | A back vector is created with SpeI and PstI to insert a donor fragment after a component on a vector. This vector is created: |
Revision as of 23:24, 21 January 2014
Contents |
What are BioBricks, and what is their purpose?
A BioBrick is a sequence of DNA with a predefined structure and function. The information is held in a plasmid, or a circular piece of DNA that can be inserted, and replicated into bacteria.
Before BioBricks, there was little standardization in assembly techniques for DNA sequences. It was necessary to create a standardized system of biological building blocks to enable scientists to go beyond the experiment of DNA assembly and allow them to focus on more complicated research. BioBricks were created as a set of standard and interchangeable “parts” that could be assembled into sub-components. According to the Registry of Standard Biological Parts, which contains DNA for thousands of previously created parts submitted by iGEM members, a standard biological part is “a functional unit of DNA that encodes for a specific biological function. Parts have been standardized so they can be used to efficiently develop biological systems in living cells." The advantage of BioBricks is that the assembly of these parts could be outsourced to others and new experimentation could rely heavily on previously manufactured components. The BioBricks foundation was co-founded in 2005 by Drew Endy. Tom Knight and Drew Endy also helped co-found the Registry of Standard Biological Parts.[1]
The creation of a standard sequence interface for all BioBricks:
The original BioBrick assembly standard created by Tom Knight:
In order to easily joint and manipulate segments of DNA, BioBrick assembly standards requires the use of defined prefix and suffix sequences that flank both sides of the BioBrick. These prefix and suffix regions (such as EcoRI, NotI, XbaI, SpeI, and PtsI) can be cut by specific restriction endonucleases. Restriction endonucleases are a key element in the cutting and conjoining of segments of DNA, and will be elaborated in further detail in a later section. (insert hyperlink here.. ask dennis how to link it to restriction endonuclease section)
The upstream end (Prefix) contains vector insert restriction sites with the following sequence:
5' --gca GAATTC GCGGCCGC T TCTAGA G --- 3' 3' --cgt CTTAAG CGCCGGCG A ACATCT C --- 5' EcoRI NotI XbaI
The downstream end (Suffix) contains vector insert restriction sites with the following sequence:
5' --- T ACTAGT A GCGGCCG CTGCAG gct--- 3' 3' --- A TGATCA T CGCCGGC GACGTC cga--- 5' SpeI NotI PstI
When combined, the entire component vector looks like this:
5’ --gca GAATTC GCGGCCGC T TCTAGA --insert--T ACTAGT A GCGGCCG CTGCAG gct-- 3' --cgt CTTAAG CGCCGGCG A ACATCT -------- A TGATCA T CGCCGGC GACGTC gca-- EcoRI NotI XbaI SpeI NotI PstI
The component vector (the insert) cannot contain any of the restriction sites to avoid any unwanted cutting by restriction enzymes, and any such sites are removed by point mutations.
Restriction Endonucleases
Restriction enzymes, or restriction endonucleases are enzymes that cleave DNA at restriction sites. A restriction site is a sequence of base pairs (generally 4-8 base pairs in length) that is recognized and spliced by a restriction enzyme. To cut DNA, restriction enzymes make two incisions: once through each side of the sugar-phosphate backbone. There are 5 kinds of restriction enzymes.
Types of Restriction Endonucleases
Type I: These were the first type of enzymes to be discovered, but have very little use in synthetic biology because they randomly cleave the DNA varying distances from the restriction site. The recognition site is composed of two different, asymmetrical portions: one containing 3-4 nucleotides, and another containing 4-5 nucleotides, and are separated by a non-specific spacer that is composed of 6-8 nucleotides. They are a multifunctional protein with both methylase and restriction activity.
Type II: These are the most commonly available and used restriction enzymes. They are a homodimer, meaning that they are composed of two identical molecules. They do not use ATP for their activity, but rather they generally utilize magnesium. Type II restriction enzymes recognize palindromic sequences, which are sequences that are read the same 5’ as they are 3’. New enzymes from this family have been discovered, but do not follow all classical criteria of type II enzymes. Subgroups have been created to categorize the enzymes that deviate from the criteria. Type II enzymes are a single function restriction enzyme and solely cleave but do not methylate.
Type III:
Considerations in the selection of Restriction Enzymes
List of Restriction Enzymes used with BioBricks
Enzyme | Source | Recognition Sequence | Cut |
---|---|---|---|
EcoRI | Escherichia coli |
5'GAATTC 3'CTTAAG |
5'---G AATTC---3' 3'---CTTAA G---5' |
NotI | Nocardia otitidis |
5'GCGGCCGC 3'CGCCGGCG |
5'---GC GGCCGC---3' 3'---CGCCGG CG---5' |
SpeI | Sphaerotilus natans |
5'ACTAGT 3'TGATCA |
5'---A CTAGT---3' 3'---TGATC A---5' |
XbaI | Xanthomonas badrii |
5'TCTAGA 3'AGATCT |
5'---T CTAGA---3' 3'---AGATC T---5' |
PstI | Providencia stuartii |
5'CTGCAG 3'GACGTC |
5'---CTGCA G---3' 3'---G ACGTC---5' |
BioBrick composition techniques
There are two methods of composing a biobrick component: Prefixing one component to another, or postfixing one component with another. Both techniques result in a new, compound component, which can then be used as either an insert or vector in other reactions.
Prefixing a recipient vector with a donor component
In order to prefix a recipient component, a front vector is created. To do so, the component is cut with EcoRI and XbaI enzymes:
5' --gca G *CTAGA G---- 3' 3' --cgt CTTAA* T C---- 5' EcoRI XbaI
EcoRI and SpeI are used to cut the donor component, creating a front insert:
5' *AATTC GCGGCCGC T TCTAGA G --Insert-- T A 3' 3' G CGCCGGCG A ACATCT C --Insert-- A TGATC* 5' EcoRI NotI XbaI SpeI
The EcoRI sites and mixed SpeI/XbaI sites are ligated creating this segment:
5' --gca G *AATTC GCGGCCGC T TCTAGA G--insert--T A *CTAGA G---- 3' 3' --cgt CTTAA* G CGCCGGCG A ACATCT C--insert--A TGATC* T C---- 5' EcoRI NotI XbaI Mixed
The EcoRI and XbaI sites are recreated, and a mixed, uncuttable SpeI/XbaI site is created. The other restriction regions SpeI and PstI remain untouched.
Postfixing a recipient vector with a donor component
A back vector is created with SpeI and PstI to insert a donor fragment after a component on a vector. This vector is created:
5' --T A *G gct--- 3' 3' --A TGATC* ACGTC cga--- 5' SpeI PstI
The donor component is cut with XbaI and PstI to create a back insert:
5' *CTAGA G --insert-- T ACTAGT A GCGGCCG CTGCA 3' 3' T C --insert-- A TGATCA T CGCCGGC G* 5' XbaI SpeI NotI PstI
Similar to the Prefixing method, the two products are ligated together. The SpeI and PstI sites are restored, while there is an uncuttable, mixed SpeI/XbaI site that is made at the junction of the two inserts. In contrast to the Prefixing method, now the EcoRI and XbaI sites remain untouched in the upstream region:
5' --T A *CTAGA G --insert-- T ACTAGT A GCGGCCG CTGCA *G gct--- 3' 3' --A TGATC* T C --insert-- A TGATCA T CGCCGGC G* ACGTC cga--- 5' Mixed SpeI NotI PstI