"

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-	<pre>	+	'''BBF RFC xx: "BioSandwich" - a homology based assembly method using a library
-	BBF RFC xx: "BioSandwich" - a homology based assembly method using a library	+	of standard parts'''
-	of standard parts	+
	Chris French, Allan Crossman		Chris French, Allan Crossman
	September, 2011		September, 2011
-	1. Purpose	+	==1. Purpose==
	This Request for Comments (RFC) describes a strategy for using homology-based		This Request for Comments (RFC) describes a strategy for using homology-based
	assembly methods to assemble parts from a library in any order.		assembly methods to assemble parts from a library in any order.
-	2. Relation to other BBF RFCs	+	==2. Relation to other BBF RFCs==
	BBF RFC xx does not update or replace any earlier BBF RFC.		BBF RFC xx does not update or replace any earlier BBF RFC.
-	3. Copyright Notice	+	==3. Copyright Notice==
	Copyright (C) The BioBricks Foundation (2011). All Rights Reserved.		Copyright (C) The BioBricks Foundation (2011). All Rights Reserved.
-	4. Background	+	==4. Background==
	One of the main aspects of synthetic biology is the assembly of standard		One of the main aspects of synthetic biology is the assembly of standard
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	different combinations of the same parts in different orders.		different combinations of the same parts in different orders.
-	5. Outline	+	==5. Outline==
	BioSandwich parts are reusable because they come in a standard format (much		BioSandwich parts are reusable because they come in a standard format (much
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	"spacers"). These spacers serve two purposes:		"spacers"). These spacers serve two purposes:
-	* They create homology between the end of one part and the start of	+	* They create homology between the end of one part and the start of
	another; this allows homology-based assembly.		another; this allows homology-based assembly.
-	* They can incorporate short meaningful sequences such as ribosome binding	+	* They can incorporate short meaningful sequences such as ribosome binding
	sites, linkers for fusion proteins, etc.		sites, linkers for fusion proteins, etc.
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	determine the order of the parts in the final assembly.		determine the order of the parts in the final assembly.
-	6. Formats	+	==6. Formats==
-	6.1. Normal Parts	+	===6.1. Normal Parts===
	Parts must be free of internal BglII restriction sites (agatct) and SpeI sites		Parts must be free of internal BglII restriction sites (agatct) and SpeI sites
	(actagt). Each part must be made with a BglII site at the start, and a SpeI		(actagt). Each part must be made with a BglII site at the start, and a SpeI
	site at the end. If compliance with RFC10 is desired, the full format becomes:		site at the end. If compliance with RFC10 is desired, the full format becomes:
-		+	<pre>
	gaattcgcggccgcttctagagatct NNN NNN NNN NNt actagtagcggccgctgcag		gaattcgcggccgcttctagagatct NNN NNN NNN NNt actagtagcggccgctgcag
-		+	</pre>
	After cutting with BglII and SpeI, we have the following (shown in frame,		After cutting with BglII and SpeI, we have the following (shown in frame,
	which is relevant for fusion proteins):		which is relevant for fusion proteins):
-		+	<pre>
	5'    ga tct NNN NNN NNN NNt a        3'		5'    ga tct NNN NNN NNN NNt a        3'
	3'        a nnn nnn nnn nna tga tc    5'		3'        a nnn nnn nnn nna tga tc    5'
-		+	</pre>
	When a part is used for fusion proteins, the "t" base at the start of the		When a part is used for fusion proteins, the "t" base at the start of the
	RFC10 suffix becomes the final base of the final codon in the part. Design of		RFC10 suffix becomes the final base of the final codon in the part. Design of
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	required, this "t" base can be replaced with anything.)		required, this "t" base can be replaced with anything.)
-	6.2. Normal Spacers	+	===6.2. Normal Spacers===
	Spacers are designed as a set of oligonucleotides that can be attached to any		Spacers are designed as a set of oligonucleotides that can be attached to any
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	The format for the upstream spacers is as follows (both strands shown):		The format for the upstream spacers is as follows (both strands shown):
-		+	<pre>
	5'    ct agc NNN NNN NNN g        3'              (forward spoligo)		5'    ct agc NNN NNN NNN g        3'              (forward spoligo)
	3'    ga tcg nnn nnn nnn cct ag    5'              (reverse spoligo ONE)		3'    ga tcg nnn nnn nnn cct ag    5'              (reverse spoligo ONE)
-		+	</pre>
	The format for the downstream spacers is:		The format for the downstream spacers is:
-		+	<pre>
	5'    ct agc NNN NNN NNN g        3'              (forward spoligo)		5'    ct agc NNN NNN NNN g        3'              (forward spoligo)
	3'        g nnn nnn nnn c        5'              (reverse spoligo TWO)		3'        g nnn nnn nnn c        5'              (reverse spoligo TWO)
-		+	</pre>
	Note that the forward spoligo is used for both upstream and downstream		Note that the forward spoligo is used for both upstream and downstream
	attachment.		attachment.
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	are to be used after a coding part which lacks one.		are to be used after a coding part which lacks one.
-	6.3 Vector Part	+	===6.3 Vector Part===
	A vector is made as a PCR product with a BglII site (agatct) at the 5' end and		A vector is made as a PCR product with a BglII site (agatct) at the 5' end and
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	If one wishes the final products (after insertion into the vector) to be		If one wishes the final products (after insertion into the vector) to be
	RFC10-compliant, the format for a vector is as follows:		RFC10-compliant, the format for a vector is as follows:
-		+	<pre>
	agatct [nnn] tactagtagcggccgctgcag [ori, cmlR, etc] gaattcgcggccgcttctaga		agatct [nnn] tactagtagcggccgctgcag [ori, cmlR, etc] gaattcgcggccgcttctaga
-		+	</pre>
	Note that a few additional bases must also be present at the 5' and 3' ends to		Note that a few additional bases must also be present at the 5' and 3' ends to
	allow the restriction enzymes space to work. When spacers are to be annealed,		allow the restriction enzymes space to work. When spacers are to be annealed,
	the vector is cut with BglII and XbaI (not SpeI).		the vector is cut with BglII and XbaI (not SpeI).
-	6.4. Start Spacer	+	===6.4. Start Spacer===
	For compliance with RFC10, the spacer that connects the vector to the first		For compliance with RFC10, the spacer that connects the vector to the first
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	Spoligos attaching upstream of first part:		Spoligos attaching upstream of first part:
-		+	<pre>
	5'    ctagag NNNNNNNN g        3'              (forward spoligo)		5'    ctagag NNNNNNNN g        3'              (forward spoligo)
	3'    gatctc nnnnnnnn cctag    5'              (reverse spoligo ONE)		3'    gatctc nnnnnnnn cctag    5'              (reverse spoligo ONE)
-		+	</pre>
	Spoligos attaching downstream of vector:		Spoligos attaching downstream of vector:
-		+	<pre>
	5'    ctagag NNNNNNNN g        3'              (forward spoligo)		5'    ctagag NNNNNNNN g        3'              (forward spoligo)
	3'        tc nnnnnnnn c        5'              (reverse spoligo TWO)		3'        tc nnnnnnnn c        5'              (reverse spoligo TWO)
-		+	</pre>
	This format ensures that the final product contains a standard RFC10 prefix		This format ensures that the final product contains a standard RFC10 prefix
	and suffix.		and suffix.
-	7. Assembly	+	==7. Assembly==
-	7.1. Digestion of Parts and Vector	+	===7.1. Digestion of Parts and Vector===
	Each part must be digested separately.		Each part must be digested separately.
-	* Parts: digest with BglII and SpeI in NEB Buffer 2	+	* Parts: digest with BglII and SpeI in NEB Buffer 2
-	* Vector: digest with BglII and XbaI in NEB Buffer 2 or 3	+	* Vector: digest with BglII and XbaI in NEB Buffer 2 or 3
	Each tube gets:		Each tube gets:
-		+	<pre>
	36 uL    Water		36 uL    Water
	5 uL    DNA		5 uL    DNA
Line 159:		Line 158:
	2 uL    Enzyme 1		2 uL    Enzyme 1
	2 uL    Enzyme 2		2 uL    Enzyme 2
-		+	</pre>
	The digestions are then left for 2 hours at 37 C. Afterwards they are purified		The digestions are then left for 2 hours at 37 C. Afterwards they are purified
	with 5 uL glass beads, and eluted to 10 uL EB (protocol).		with 5 uL glass beads, and eluted to 10 uL EB (protocol).
-	7.2. Ligation of Parts and Vector to Spacers	+	===7.2. Ligation of Parts and Vector to Spacers===
	The parts and vector must now be ligated to the correct spacers, as follows.		The parts and vector must now be ligated to the correct spacers, as follows.
	Each tube gets:		Each tube gets:
-		+	<pre>
	10 uL    Water		10 uL    Water
	5 uL    DNA		5 uL    DNA
Line 175:		Line 174:
	2 uL    Ligase buffer		2 uL    Ligase buffer
	1 uL    T4 DNA ligase		1 uL    T4 DNA ligase
-		+	</pre>
	Tubes should then be left for 9 hours at 16 C. They must then be purified.		Tubes should then be left for 9 hours at 16 C. They must then be purified.
-	7.3. Homology-Based Assembly	+	===7.3. Homology-Based Assembly===
	The parts can now be assembled using a number of different homology-based		The parts can now be assembled using a number of different homology-based
Line 184:		Line 183:
	Polymerase Extension Cloning, and others.		Polymerase Extension Cloning, and others.
-	8. Authors' contact information	+	==8. Authors' contact information==
-	Chris French: c.french@ed.ac.uk	+	:Chris French: c.french@ed.ac.uk
-	Allan Crossman: igem@nothinginbiology.com	+	:Allan Crossman: igem@nothinginbiology.com
-	References	+	==References==
	Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA, Smith HO (2009)		Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA, Smith HO (2009)
	Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature		Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature
	Methods 6:343-345 (doi: 10.1038/nmeth.1318).		Methods 6:343-345 (doi: 10.1038/nmeth.1318).
-	</pre>

---

Revision as of 10:51, 7 September 2011

BBF RFC xx: "BioSandwich" - a homology based assembly method using a library of standard parts

Chris French, Allan Crossman September, 2011

Contents 1 1. Purpose 2 2. Relation to other BBF RFCs 3 3. Copyright Notice 4 4. Background 5 5. Outline 6 6. Formats 6.1 6.1. Normal Parts 6.2 6.2. Normal Spacers 6.3 6.3 Vector Part 6.4 6.4. Start Spacer 7 7. Assembly 7.1 7.1. Digestion of Parts and Vector 7.2 7.2. Ligation of Parts and Vector to Spacers 7.3 7.3. Homology-Based Assembly 8 8. Authors' contact information 9 References

1. Purpose

This Request for Comments (RFC) describes a strategy for using homology-based assembly methods to assemble parts from a library in any order.

2. Relation to other BBF RFCs

BBF RFC xx does not update or replace any earlier BBF RFC.

3. Copyright Notice

Copyright (C) The BioBricks Foundation (2011). All Rights Reserved.

4. Background

One of the main aspects of synthetic biology is the assembly of standard modular parts from a library to produce larger constructs. There are two main types of assembly strategy: hierarchical and homology-based.

The RFC10 format, which is standard for iGEM projects, is based on heirarchical assembly methods such as Standard Assembly and Three Antibiotic Assembly. The main advantage of RFC10 is flexibility: any combination of parts can be assembled in any order. The main disadvantage is speed.

Many iGEM teams now use homology-based assembly strategies, such as Gibson Assembly. These methods use PCR to create parts that have homologous ends. They are then assembled using the method of Gibson et al (2009). This method is relatively fast. However, the order of the parts in the final construct is entirely determined by the primers chosen for the initial PCR reactions. If a different order is desired, parts have to be remade with new PCR primers.

BioSandwich is a hybrid that combines many of the benefits of hierarchical assembly and homology-based assembly. In particular, it is useful for creating different combinations of the same parts in different orders.

5. Outline

BioSandwich parts are reusable because they come in a standard format (much like ordinary BioBricks) with restriction sites flanking the part. The restriction sites are BglII (agatct) in the prefix, and SpeI (actagt) in the suffix. However, these restriction sites are not used directly for assembly; instead they are used to attach short (~35 bp) oligonucleotides (hereafter "spacers"). These spacers serve two purposes:

• They create homology between the end of one part and the start of

another; this allows homology-based assembly.

• They can incorporate short meaningful sequences such as ribosome binding

sites, linkers for fusion proteins, etc.

Any lab using BioSandwich will want to keep a small library of different spacers for different purposes. Once (carefully chosen) spacers have been attached to each part, homology based assembly can be carried out in a single reaction. Precisely which spacers have been attached to which parts will determine the order of the parts in the final assembly.

6. Formats

6.1. Normal Parts

Parts must be free of internal BglII restriction sites (agatct) and SpeI sites (actagt). Each part must be made with a BglII site at the start, and a SpeI site at the end. If compliance with RFC10 is desired, the full format becomes:

	gaattcgcggccgcttctagagatct NNN NNN NNN NNt actagtagcggccgctgcag

After cutting with BglII and SpeI, we have the following (shown in frame, which is relevant for fusion proteins):

	5'    ga tct NNN NNN NNN NNt a         3'
	3'         a nnn nnn nnn nna tga tc    5'

When a part is used for fusion proteins, the "t" base at the start of the RFC10 suffix becomes the final base of the final codon in the part. Design of the part should take this into account. This is often done by making this final codon GGT, coding for an innocuous glycine residue. (If a fusion protein is not being made, no such consideration is needed. If RFC10-compliance is not required, this "t" base can be replaced with anything.)

6.2. Normal Spacers

Spacers are designed as a set of oligonucleotides that can be attached to any part via the part's restriction sites. Each type of spacer will be attached upstream of one part and downstream of another. It is necessary to create three different single-stranded oligonucleotides (spacer oligos or "spoligos") for each spacer so as to allow annealing and ligation at the restriction sites. They should be designed so that the spacer's non-ligating ends are blunt.

The format for the upstream spacers is as follows (both strands shown):

	5'    ct agc NNN NNN NNN g         3'               (forward spoligo)
	3'    ga tcg nnn nnn nnn cct ag    5'               (reverse spoligo ONE)

The format for the downstream spacers is:

	5'    ct agc NNN NNN NNN g         3'               (forward spoligo)
	3'         g nnn nnn nnn c         5'               (reverse spoligo TWO)

Note that the forward spoligo is used for both upstream and downstream attachment.

The content of different spacers should be varied to avoid homology. It is recommended that non-coding spacers have an in-frame stop codon, in case they are to be used after a coding part which lacks one.

6.3 Vector Part

A vector is made as a PCR product with a BglII site (agatct) at the 5' end and an XbaI site (tctaga) at the 3' end. Since XbaI produces sticky-ends compatible with SpeI, the vector is compatible with standard spacers.

If one wishes the final products (after insertion into the vector) to be RFC10-compliant, the format for a vector is as follows:

	agatct [nnn] tactagtagcggccgctgcag [ori, cmlR, etc] gaattcgcggccgcttctaga

Note that a few additional bases must also be present at the 5' and 3' ends to allow the restriction enzymes space to work. When spacers are to be annealed, the vector is cut with BglII and XbaI (not SpeI).

6.4. Start Spacer

For compliance with RFC10, the spacer that connects the vector to the first part must be in the following format:

Spoligos attaching upstream of first part:

	5'    ctagag NNNNNNNN g         3'               (forward spoligo)
	3'    gatctc nnnnnnnn cctag     5'               (reverse spoligo ONE)

Spoligos attaching downstream of vector:

	5'    ctagag NNNNNNNN g         3'               (forward spoligo)
	3'        tc nnnnnnnn c         5'               (reverse spoligo TWO)

This format ensures that the final product contains a standard RFC10 prefix and suffix.

7. Assembly

7.1. Digestion of Parts and Vector

Each part must be digested separately.

• Parts: digest with BglII and SpeI in NEB Buffer 2
• Vector: digest with BglII and XbaI in NEB Buffer 2 or 3

Each tube gets:

	36 uL     Water
	 5 uL     DNA
	 5 uL     Buffer
	 2 uL     Enzyme 1
	 2 uL     Enzyme 2

The digestions are then left for 2 hours at 37 C. Afterwards they are purified with 5 uL glass beads, and eluted to 10 uL EB (protocol).

7.2. Ligation of Parts and Vector to Spacers

The parts and vector must now be ligated to the correct spacers, as follows.

Each tube gets:

	10 uL     Water
	 5 uL     DNA
	 1 uL     Spacer pair #1
	 1 uL     Spacer pair #2
	 2 uL     Ligase buffer
	 1 uL     T4 DNA ligase

Tubes should then be left for 9 hours at 16 C. They must then be purified.

7.3. Homology-Based Assembly

The parts can now be assembled using a number of different homology-based assembly methods, including Gibson Assembly, Overlap Extension PCR, Circular Polymerase Extension Cloning, and others.

8. Authors' contact information

Chris French: c.french@ed.ac.uk

Allan Crossman: igem@nothinginbiology.com

References

Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA, Smith HO (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods 6:343-345 (doi: 10.1038/nmeth.1318).