Richard Lab:Bio-Brick Formats - Revision history

Michael A. Speer: /* Introduction */

2011-03-24T14:14:44Z

Introduction

←Older revision		Revision as of 14:14, 24 March 2011
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	==Introduction==		==Introduction==
-	In general the Richard Lab uses the standard [http://partsregistry.org/Help:BioBrick_Prefix_and_Suffix Bio-Brick] format. However occasionally this format is incompatible with our goals (e.g. making protein fusions) or is unnecessarilly cumbersome (e.g. PCR overlap extension). To have a review on why (and how) we use these sites see the our [[Richard Lab:~~Bio-Brick Assembly Schedule\|New Standard~~ Assembly]] page.	+	In general the Richard Lab uses the standard [http://partsregistry.org/Help:BioBrick_Prefix_and_Suffix Bio-Brick] format. However occasionally this format is incompatible with our goals (e.g. making protein fusions) or is unnecessarilly cumbersome (e.g. PCR overlap extension). To have a review on why (and how) we use these sites see the our [[Richard Lab:Amplified Insert Assembly]] page.

	==Formats==		==Formats==

Michael A. Speer: /* Notes */

2011-03-24T14:13:53Z

Notes

←Older revision		Revision as of 14:13, 24 March 2011
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	*......AAGCTTtACTAGTagcggcgCTGCAG		*......AAGCTTtACTAGTagcggcgCTGCAG
	====Notes====		====Notes====
-	*Notice that the HindIII restriction sites are "inside" of the normal bio-brick sites, and in the case of the prefix the start codon is between the fusion site and the biobrick sites. This allows us to use these proteins individually or fused in normal bio-brick assembly.	+	*Notice that the HindIII restriction sites are "inside" of the normal bio-brick sites, and in the case of the prefix the start codon is between the fusion site and the biobrick sites. This allows us to use these proteins individually or fused in normal bio-brick assembly.
		+	*Don't use BamHI if you don't have to. It's not able to be heat inactivated.
	*One major limitation of this method is that the proteins are determined N terminus or C terminus fusions. In other words, you can take an N terminus signal peptide (with HindIII site in the suffix) and fuse it behind a C terminus enzyme (with HindIII site in the prefix). Since most of the time we're only dealing with signal peptides this usually doesn't matter.		*One major limitation of this method is that the proteins are determined N terminus or C terminus fusions. In other words, you can take an N terminus signal peptide (with HindIII site in the suffix) and fuse it behind a C terminus enzyme (with HindIII site in the prefix). Since most of the time we're only dealing with signal peptides this usually doesn't matter.
	*If you want, simply replace the HindIII site with a BamHI site and you can avoid any cloning difficulty due to unwanted sites in your vector or coding sequence.		*If you want, simply replace the HindIII site with a BamHI site and you can avoid any cloning difficulty due to unwanted sites in your vector or coding sequence.

Michael A. Speer: /* Protein Fusions */

2011-01-28T19:20:01Z

Protein Fusions

←Older revision		Revision as of 19:20, 28 January 2011
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	===Protein Fusions===		===Protein Fusions===
-	From time to time it becomes necessary to make protein fusions. As This is not a common occurance we use a really simple method which is not really bio-bricking. we either use HindIII (AAGCTT) or BamHI (GGATCC) as in frame restriction sites which couple with identical in frame sites in another protein. Mostly this involves attaching signal peptides to the N terminus of enzymes. In this case the modified biobrick prefix for an enzyme is:	+	From time to time it becomes necessary to make protein fusions. As This is not a common occurance we use a really simple method which is not really bio-bricking. We either use HindIII (AAGCTT) or BamHI (GGATCC) as in frame restriction sites which couple with identical in frame sites in another protein. Mostly this involves attaching signal peptides to the N terminus of enzymes. In this case the modified biobrick prefix for an enzyme is:
	*GAATTCgcggccgctTCTAGAtgAAGCTT......		*GAATTCgcggccgctTCTAGAtgAAGCTT......
	and the modified suffix for the signal peptide is:		and the modified suffix for the signal peptide is:

Michael A. Speer: /* Notes */

2011-01-25T19:08:25Z

Notes

←Older revision		Revision as of 19:08, 25 January 2011
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	*One major limitation of this method is that the proteins are determined N terminus or C terminus fusions. In other words, you can take an N terminus signal peptide (with HindIII site in the suffix) and fuse it behind a C terminus enzyme (with HindIII site in the prefix). Since most of the time we're only dealing with signal peptides this usually doesn't matter.		*One major limitation of this method is that the proteins are determined N terminus or C terminus fusions. In other words, you can take an N terminus signal peptide (with HindIII site in the suffix) and fuse it behind a C terminus enzyme (with HindIII site in the prefix). Since most of the time we're only dealing with signal peptides this usually doesn't matter.
	*If you want, simply replace the HindIII site with a BamHI site and you can avoid any cloning difficulty due to unwanted sites in your vector or coding sequence.		*If you want, simply replace the HindIII site with a BamHI site and you can avoid any cloning difficulty due to unwanted sites in your vector or coding sequence.
-	*This also allows you to really simplify your life when cloning coding sequences via PCR. Instead of having a complicated job extending the sequence to include the entire bio-brick prefix or suffix, just add the HindIII site and the SpeI site (for an enzyme) or the HindIII site and the XbaI site (for a signal peptide) and then drop it in place of a similar part. the prefix and suffix will fall into place.	+	*This also allows you to really simplify your life when cloning coding sequences via PCR. Instead of having a complicated job extending the sequence to include the entire bio-brick prefix or suffix, just add the HindIII site and the SpeI site (for an enzyme) or the HindIII site and the XbaI site (for a signal peptide) and then drop it in place of a similar part. The rest of the prefix and suffix will then fall into place.


	Back to [[Richard_Lab:protocols \| Protocols]]		Back to [[Richard_Lab:protocols \| Protocols]]

Michael A. Speer: /* Notes */

2011-01-25T19:07:31Z

Notes

←Older revision		Revision as of 19:07, 25 January 2011
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	====Notes====		====Notes====
	*Notice that the HindIII restriction sites are "inside" of the normal bio-brick sites, and in the case of the prefix the start codon is between the fusion site and the biobrick sites. This allows us to use these proteins individually or fused in normal bio-brick assembly.		*Notice that the HindIII restriction sites are "inside" of the normal bio-brick sites, and in the case of the prefix the start codon is between the fusion site and the biobrick sites. This allows us to use these proteins individually or fused in normal bio-brick assembly.
-	*One major limitation of this method is that the proteins are determined N terminus or C terminus fusions. In other words, you can take an N terminus signal peptide (with HindIII site in the suffix) and fuse it behind a C terminus enzyme (with HindIII site in the prefix). Since most of the time we're ~~onoy~~ dealing with signal peptides this usually doesn't matter.	+	*One major limitation of this method is that the proteins are determined N terminus or C terminus fusions. In other words, you can take an N terminus signal peptide (with HindIII site in the suffix) and fuse it behind a C terminus enzyme (with HindIII site in the prefix). Since most of the time we're only dealing with signal peptides this usually doesn't matter.
	*If you want, simply replace the HindIII site with a BamHI site and you can avoid any cloning difficulty due to unwanted sites in your vector or coding sequence.		*If you want, simply replace the HindIII site with a BamHI site and you can avoid any cloning difficulty due to unwanted sites in your vector or coding sequence.
	*This also allows you to really simplify your life when cloning coding sequences via PCR. Instead of having a complicated job extending the sequence to include the entire bio-brick prefix or suffix, just add the HindIII site and the SpeI site (for an enzyme) or the HindIII site and the XbaI site (for a signal peptide) and then drop it in place of a similar part. the prefix and suffix will fall into place.		*This also allows you to really simplify your life when cloning coding sequences via PCR. Instead of having a complicated job extending the sequence to include the entire bio-brick prefix or suffix, just add the HindIII site and the SpeI site (for an enzyme) or the HindIII site and the XbaI site (for a signal peptide) and then drop it in place of a similar part. the prefix and suffix will fall into place.

Michael A. Speer: /* Short Biobrick */

2011-01-25T00:07:43Z

Short Biobrick

←Older revision		Revision as of 00:07, 25 January 2011
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	===Short Biobrick===		===Short Biobrick===
-	Because the NotI sites in the biobricks are rarely used a shortened biobrick system can be employed. This is especailly usefull when adding "flanking" restriction sites to sequences via overlap extention PCR. The shortened sequences are thus:	+	Because the NotI sites in the biobricks are rarely used, a shortened biobrick system can be employed. This is especailly usefull when adding "flanking" restriction sites to sequences via overlap extention PCR. The shortened sequences are thus:
	*Prefix		*Prefix
	::*GAATCgctTCTAGAg		::*GAATCgctTCTAGAg

Michael A. Speer: /* Protein Fusions */

2011-01-25T00:07:09Z

Protein Fusions

←Older revision		Revision as of 00:07, 25 January 2011
Line 36:		Line 36:
	*GAATTCgcggccgctTCTAGAtgAAGCTT......		*GAATTCgcggccgctTCTAGAtgAAGCTT......
	and the modified suffix for the signal peptide is:		and the modified suffix for the signal peptide is:
-	*AAGCTTtACTAGTagcggcgCTGCAG	+	*......AAGCTTtACTAGTagcggcgCTGCAG
	====Notes====		====Notes====
	*Notice that the HindIII restriction sites are "inside" of the normal bio-brick sites, and in the case of the prefix the start codon is between the fusion site and the biobrick sites. This allows us to use these proteins individually or fused in normal bio-brick assembly.		*Notice that the HindIII restriction sites are "inside" of the normal bio-brick sites, and in the case of the prefix the start codon is between the fusion site and the biobrick sites. This allows us to use these proteins individually or fused in normal bio-brick assembly.

Michael A. Speer: /* Protein Fusions */

2011-01-25T00:06:39Z

Protein Fusions

←Older revision		Revision as of 00:06, 25 January 2011
Line 37:		Line 37:
	and the modified suffix for the signal peptide is:		and the modified suffix for the signal peptide is:
	*AAGCTTtACTAGTagcggcgCTGCAG		*AAGCTTtACTAGTagcggcgCTGCAG
-	Notice that the HindIII restriction sites are "inside" of the normal bio-brick sites, and in the case of the prefix the start codon is between the fusion site and the biobrick sites. This allows us to use these proteins individually or fused in normal bio-brick assembly.	+	====Notes====
-	One major limitation of this method is that the proteins are determined N terminus or C terminus fusions. In other words, you can take an N terminus signal peptide (with HindIII site in the suffix) and fuse it behind a C terminus enzyme (with HindIII site in the prefix.	+	*Notice that the HindIII restriction sites are "inside" of the normal bio-brick sites, and in the case of the prefix the start codon is between the fusion site and the biobrick sites. This allows us to use these proteins individually or fused in normal bio-brick assembly.
-	If you want simply replace the HindIII site with a BamHI site and you can avoid any cloning difficulty due to unwanted sites in your vector or coding sequence.	+	*One major limitation of this method is that the proteins are determined N terminus or C terminus fusions. In other words, you can take an N terminus signal peptide (with HindIII site in the suffix) and fuse it behind a C terminus enzyme (with HindIII site in the prefix). Since most of the time we're onoy dealing with signal peptides this usually doesn't matter.
-		+	*If you want, simply replace the HindIII site with a BamHI site and you can avoid any cloning difficulty due to unwanted sites in your vector or coding sequence.
-	This also allows you to really simplify your life when cloning coding sequences via PCR. Instead of having a complicated ~~time~~ extending the sequence to include the entire bio-brick prefix or suffix, just add the	+	*This also allows you to really simplify your life when cloning coding sequences via PCR. Instead of having a complicated job extending the sequence to include the entire bio-brick prefix or suffix, just add the HindIII site and the SpeI site (for an enzyme) or the HindIII site and the XbaI site (for a signal peptide) and then drop it in place of a similar part. the prefix and suffix will fall into place.


	Back to [[Richard_Lab:protocols \| Protocols]]		Back to [[Richard_Lab:protocols \| Protocols]]

Michael A. Speer: /* Protein Fusions */

2011-01-25T00:02:10Z

Protein Fusions

←Older revision		Revision as of 00:02, 25 January 2011
Line 33:		Line 33:

	===Protein Fusions===		===Protein Fusions===
		+	From time to time it becomes necessary to make protein fusions. As This is not a common occurance we use a really simple method which is not really bio-bricking. we either use HindIII (AAGCTT) or BamHI (GGATCC) as in frame restriction sites which couple with identical in frame sites in another protein. Mostly this involves attaching signal peptides to the N terminus of enzymes. In this case the modified biobrick prefix for an enzyme is:
		+	*GAATTCgcggccgctTCTAGAtgAAGCTT......
		+	and the modified suffix for the signal peptide is:
		+	*AAGCTTtACTAGTagcggcgCTGCAG
		+	Notice that the HindIII restriction sites are "inside" of the normal bio-brick sites, and in the case of the prefix the start codon is between the fusion site and the biobrick sites. This allows us to use these proteins individually or fused in normal bio-brick assembly.
		+	One major limitation of this method is that the proteins are determined N terminus or C terminus fusions. In other words, you can take an N terminus signal peptide (with HindIII site in the suffix) and fuse it behind a C terminus enzyme (with HindIII site in the prefix.
		+	If you want simply replace the HindIII site with a BamHI site and you can avoid any cloning difficulty due to unwanted sites in your vector or coding sequence.

-		+	This also allows you to really simplify your life when cloning coding sequences via PCR. Instead of having a complicated time extending the sequence to include the entire bio-brick prefix or suffix, just add the


	Back to [[Richard_Lab:protocols \| Protocols]]		Back to [[Richard_Lab:protocols \| Protocols]]

Michael A. Speer: /* Short Biobrick */

2011-01-24T22:44:16Z

Short Biobrick

←Older revision		Revision as of 22:44, 24 January 2011
Line 21:		Line 21:
	===Short Biobrick===		===Short Biobrick===
	Because the NotI sites in the biobricks are rarely used a shortened biobrick system can be employed. This is especailly usefull when adding "flanking" restriction sites to sequences via overlap extention PCR. The shortened sequences are thus:		Because the NotI sites in the biobricks are rarely used a shortened biobrick system can be employed. This is especailly usefull when adding "flanking" restriction sites to sequences via overlap extention PCR. The shortened sequences are thus:
		+	*Prefix
		+	::*GAATCgctTCTAGAg
		+	::*The EcoRI and XbaI sites are in CAPS.
		+	*Protein Coding Prefix
		+	::*GAATCgctTCTAG
		+	::*using this prefix the first base of the start codon (i.e. the "A" in "ATG") will complete the XbaI site.
		+	*Suffix
		+	::*tACTAGTagctCTGCAG
		+	*Scar
		+	::*TACTAGAG for non coding parts and TACTAG between coding parts and Ribosome binding sites.

	===Protein Fusions===		===Protein Fusions===