Difference between revisions of "Assembly:RBS-CDS issues"
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'''Note: page needs reformatting'''
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[[Category:BioBrick RFC 10]]
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{{HelpPage/MainLinks}}
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== Transfer Curves ==
  
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The transfer curve of a BioBrick device is measured from input PoPs to output PoPs. For a protein-DNA inhibition system, this transfer curve depends on the rates and efficiencies of many stages in a chain that includes transcriptional efficiency, mRNA lifetime, ribosome binding efficiency, translation initiation and efficiency, protein lifetime, protein concentration, protein multimerization, protein-DNA binding rates and efficiencies, cooperative effects with other molecules, RNA polymerase effects, system copy count, and other system specific effects.
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The [http://openwetware.org/wiki/Endy:F2620/Transfer_Curve transfer curve] of a BioBrick device is measured from input PoPs to output PoPs. (More on PoPs [http://parts2.mit.edu/wiki/index.php/Abstraction_hierarchy_and_PoPS here].) For a protein-DNA inhibition system, the transfer curve depends on the rates and efficiencies of many stages in a chain that includes:
 +
 
 +
*transcriptional efficiency
 +
*mRNA lifetime
 +
*ribosome binding efficiency
 +
*translation initiation and efficiency
 +
*protein lifetime
 +
*protein concentration
 +
*protein multimerization
 +
*protein-DNA binding rates and efficiencies
 +
*cooperative effects with other molecules
 +
*RNA polymerase effects
 +
*system copy count
 +
 
 +
and more system-specific variables, few if any of which can be measured directly.
  
 
If BioBrick parts are to be interoperable, intentional engineering of this chain of events will be required to produce a desired transfer function.
 
If BioBrick parts are to be interoperable, intentional engineering of this chain of events will be required to produce a desired transfer function.
  
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Different ribosome binding sites (RBS) bind ribosomes with different efficiencies. While the overall efficiency may not be directly proportional to this efficiency, it is an important variable. In the future, we expect that this data book will have a table such as the following:
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== Ribosome Binding Sites (RBS) ==
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 +
Different RBS bind ribosomes with different efficiencies. While the overall efficiency may not be directly proportional to this efficiency, it is an important variable. In the future, we expect that this data book will have a table such as the following:
  
 
'''Standard BioBrick Ribosome Binding Sites'''
 
'''Standard BioBrick Ribosome Binding Sites'''
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A BioBrick engineer armed with this table could perform a first-order reengineering of the transfer function by measuring a component with BBa_B0031 and then selecting a more appropriate RBS.
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A BioBrick engineer armed with this table could perform a first-order reengineering of the transfer function by measuring a component with [https://parts.igem.org/wiki/index.php/Part:BBa_B0031 BBa_B0031] and then selecting a more appropriate RBS.
  
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In order to allow easy variation in the RBS, some BioBrick alpha parts have their RBS and their protein coding regions implemented in separate parts. The combined, normalized, part is also available for convenience. In order to divide the BioBrick part into its RBS and coding region, a set of BioBrick restriction sites must be designed.
+
In order to allow easy variation in the RBS, some BioBrick parts have their RBS and their protein coding regions implemented in separate parts. The combined, normalized part is also available for convenience. In order to divide the BioBrick part into its RBS and coding region, a set of BioBrick restriction sites must be designed.
  
 
== Standard Assembly and the RBS ==
 
== Standard Assembly and the RBS ==
  
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The RBS is physically near the start codon (in BioBricks, this will always be ATG). The "sequence logo" for the RBS is shown below. The size of each letter is proportional to the frequency of that base in the RBS.
+
The RBS is physically near the start codon.  (In BioBricks, this will always be ATG.)  The "sequence logo", similar to a consensus sequence, for the RBS is shown below. The size of each letter is proportional to the frequency of that base in the RBS.
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From Tom Schneider, "A Gallery of Sequence Logos"
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 +
[[Image:Ribologo-small.gif|frame|center|The RBS sequence logo, from Tom Schneider, [http://www-lmmb.ncifcrf.gov/%7Etoms/sequencelogo.html "A Gallery of Sequence Logos"]]]
 +
 
 +
This figure shows the RBS as an A/G-rich region about 10 bases upstream of the start codon. This leaves little room for a BioBrick restriction enzyme site. The standard assembly method of BioBricks normally results in an 8-base region containing a 6-base mixed SpeI/XbaI restriction site surrounded by a base on each end as shown below. If a start codon followed this sequence, the RBS would be significantly disturbed.
 +
 
 +
 
 +
 
 +
      --T ACTAGA G--
  
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This figure shows the RBS as a hump of G's or A's about 10 bases before the start codon. This leaves little room for a BioBrick restriction enzyme site. The standard assembly method of BioBricks normally results in an 8-base region containing a 6-base mixed SpeI/XbaI restriction site surrounded by a base on each end as shown below. If a start codon followed this sequence, the RBS would be significantly disturbed.
 
  
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--T ACTAGA G--
 
  
 
This problem is solved by changing the rightmost base from a G to a T. This results in the sequence shown below and limits the impact of the BioBrick overhead to six bases.
 
This problem is solved by changing the rightmost base from a G to a T. This results in the sequence shown below and limits the impact of the BioBrick overhead to six bases.
  
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Summary
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 +
[[image:RBS-CDS.gif|frame|center]]
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 +
== Summary ==
  
 
When variable RBS sequences are developed as BioBrick parts, they should have the standard BioBrick prefix and suffix as follows:
 
When variable RBS sequences are developed as BioBrick parts, they should have the standard BioBrick prefix and suffix as follows:
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Variable RBS Prefix
 
  
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GAATTC GCGGCCGC T TCTAGA G[RBS]
 
−
EcoRI  NotI      XbaI
 
  
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Variable RBS Suffix
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{| border="1" cellpadding="0"
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|-
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!width="150"|Variable RBS Prefix
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|
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    GAATTC GCGGCCGC T TCTAGA G[RBS]
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      EcoRI  NotI      XbaI
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 +
|-
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!width="150"|Variable RBS Suffix
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|
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    [RBS]T ACTAGT A GCGGCCG CTGCAG
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            SpeI    NotI    PstI
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 +
|-
 +
|}
  
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[RBS]T ACTAGT A GCGGCCG CTGCAG
 
−
      SpeI    NotI    PstI
 
  
 
When a protein coding region is designed to be preceded by a BioBrick RBS part, it should have the following prefix and suffix sequences:
 
When a protein coding region is designed to be preceded by a BioBrick RBS part, it should have the following prefix and suffix sequences:
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Protein Coding Region Prefix*
 
  
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GAATTC GCGGCCGC T TCTAG[ATG Remaining CDS]
 
−
EcoRI  NotI      XbaI
 
  
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Protein Coding Region Suffix
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{| border="1" cellpadding="0"
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|-
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!width="150"|Protein Coding Region Prefix*
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|
  
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[CDS]T ACTAGT A GCGGCCG CTGCAG
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    GAATTC GCGGCCGC T TCTAG[ATG Remaining CDS]
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      SpeI    NotI   PstI
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      EcoRI  NotI     XbaI
  
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Notes
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|-
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!width="150"|Protein Coding Region Suffix
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|
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    [CDS]T ACTAGT A GCGGCCG CTGCAG
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            SpeI    NotI    PstI
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 +
|-
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|}
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 +
 
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== Notes ==
  
 
*The only change is that the protein coding region prefix does NOT end with AGAG. instead, it ends AGATG with the ATG also being the start codon of the protein coding region.
 
*The only change is that the protein coding region prefix does NOT end with AGAG. instead, it ends AGATG with the ATG also being the start codon of the protein coding region.
  
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In BioBrick parts, we prefer using TAA as the stop codon.
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*In BioBrick parts, we prefer using TAA as the stop codon.
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<hr>
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<small>
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*Back to [[Assembly|assembly]]
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See also:
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*[[Assembly:Parts in plasmids|Parts in Plasmids]]
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*[[Assembly:Restriction enzymes|Restriction Enzymes]]
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*[[Assembly:Rolling assembly|Rolling Assembly]]
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</small>

Latest revision as of 19:51, 16 June 2017

Transfer Curves

The [http://openwetware.org/wiki/Endy:F2620/Transfer_Curve transfer curve] of a BioBrick device is measured from input PoPs to output PoPs. (More on PoPs [http://parts2.mit.edu/wiki/index.php/Abstraction_hierarchy_and_PoPS here].) For a protein-DNA inhibition system, the transfer curve depends on the rates and efficiencies of many stages in a chain that includes:

  • transcriptional efficiency
  • mRNA lifetime
  • ribosome binding efficiency
  • translation initiation and efficiency
  • protein lifetime
  • protein concentration
  • protein multimerization
  • protein-DNA binding rates and efficiencies
  • cooperative effects with other molecules
  • RNA polymerase effects
  • system copy count

and more system-specific variables, few if any of which can be measured directly.

If BioBrick parts are to be interoperable, intentional engineering of this chain of events will be required to produce a desired transfer function.

Ribosome Binding Sites (RBS)

Different RBS bind ribosomes with different efficiencies. While the overall efficiency may not be directly proportional to this efficiency, it is an important variable. In the future, we expect that this data book will have a table such as the following:

Standard BioBrick Ribosome Binding Sites

Part Number Binding Efficiency Sequence
BBa_B0030 0.6 att aaa gag gag aaa
BBa_B0031 0.07 tca cac agg aaa cc
BBa_B0032 0.3 tca cac agg aaa g


A BioBrick engineer armed with this table could perform a first-order reengineering of the transfer function by measuring a component with BBa_B0031 and then selecting a more appropriate RBS.

In order to allow easy variation in the RBS, some BioBrick parts have their RBS and their protein coding regions implemented in separate parts. The combined, normalized part is also available for convenience. In order to divide the BioBrick part into its RBS and coding region, a set of BioBrick restriction sites must be designed.

Standard Assembly and the RBS

The RBS is physically near the start codon. (In BioBricks, this will always be ATG.) The "sequence logo", similar to a consensus sequence, for the RBS is shown below. The size of each letter is proportional to the frequency of that base in the RBS.

The RBS sequence logo, from Tom Schneider, [http://www-lmmb.ncifcrf.gov/%7Etoms/sequencelogo.html "A Gallery of Sequence Logos"]

This figure shows the RBS as an A/G-rich region about 10 bases upstream of the start codon. This leaves little room for a BioBrick restriction enzyme site. The standard assembly method of BioBricks normally results in an 8-base region containing a 6-base mixed SpeI/XbaI restriction site surrounded by a base on each end as shown below. If a start codon followed this sequence, the RBS would be significantly disturbed. 


      --T ACTAGA G--


This problem is solved by changing the rightmost base from a G to a T. This results in the sequence shown below and limits the impact of the BioBrick overhead to six bases.


RBS-CDS.gif

Summary

When variable RBS sequences are developed as BioBrick parts, they should have the standard BioBrick prefix and suffix as follows:


Variable RBS Prefix 
    GAATTC GCGGCCGC T TCTAGA G[RBS]
     EcoRI  NotI      XbaI
Variable RBS Suffix 
    [RBS]T ACTAGT A GCGGCCG CTGCAG
           SpeI     NotI    PstI


When a protein coding region is designed to be preceded by a BioBrick RBS part, it should have the following prefix and suffix sequences:


Protein Coding Region Prefix* 
    GAATTC GCGGCCGC T TCTAG[ATG Remaining CDS]
     EcoRI  NotI      XbaI
Protein Coding Region Suffix 
    [CDS]T ACTAGT A GCGGCCG CTGCAG
           SpeI     NotI    PstI


Notes

  • The only change is that the protein coding region prefix does NOT end with AGAG. instead, it ends AGATG with the ATG also being the start codon of the protein coding region.
  • In BioBrick parts, we prefer using TAA as the stop codon.


See also: