Richard Lab:Bio-Brick Formats

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(Introduction)
 
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==Introduction==
==Introduction==
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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.
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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==
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===Protein Fusions===
===Protein Fusions===
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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:
*......AAGCTTtACTAGTagcggcgCTGCAG
*......AAGCTTtACTAGTagcggcgCTGCAG
====Notes====
====Notes====
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*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.

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Contents

Introduction

In general the Richard Lab uses the standard 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

Biobrick Standard

This standard uses the typical biobrick assembly enzymes [EcoRI (GAATTC), XbaI (TCTAGA), SpeI (ACTAGT), and PstI (CTGCAG)] as well as NotI sites (GCGGCCGC) flanking the part. There is a slightly different prefix for protein coding parts to put the start codon in a better spot in relation to the RBS. During assembly the XbaI site of the "rear part" and the SpeI site of the "front" part eliminate eachother and form a "scar" which forever binds the two parts together.

  • Prefix
  • GAATTCgcggccgctTCTAGAg
  • The EcoRI and XbaI sites are in CAPS while the NotI site is in bold.
  • Protein Coding Prefix
  • GAATTCgcggccgctTCTAG
  • using this prefix the first base of the start codon (i.e. the "A" in "ATG") will complete the XbaI site.
  • Suffix
  • tACTAGTagcggccgCTGCAG
  • Scar
  • Tactagag for non coding parts and Tactag between coding parts and Ribosome binding sites.

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:

  • 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

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

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.
  • 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.
  • 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 rest of the prefix and suffix will then fall into place.


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