Difference between revisions of "Ribosome Binding Sites/Prokaryotic/Constitutive/Anderson"

Line 1: Line 1:
 +
__NOTOC__
 
==Description==
 
==Description==
 
[[Image:JCA Photo.png|thumb|150px|right|The Anderson RBS family was contributed to the Registry by Prof. J. Christopher Anderson. (Photo: Peg Skorpinski)]]
 
[[Image:JCA Photo.png|thumb|150px|right|The Anderson RBS family was contributed to the Registry by Prof. J. Christopher Anderson. (Photo: Peg Skorpinski)]]

Revision as of 22:41, 6 September 2008

Description

The Anderson RBS family was contributed to the Registry by Prof. J. Christopher Anderson. (Photo: Peg Skorpinski)

The Anderson RBS family are suitable for general protein expression in E. coli. The family is known to cover a range of translation initiation rates so by testing a few family members it should be possible to find a translation initiation rate that suits your application. the family of RBS were recovered from a library screen by Chris Anderson. In the original library, 6 nucleotide locations close to where the ribosome binds were randomized (see the master sequence below). The library has not yet been quantitatively characterized (see the Characterization section below).


Obtaining members of the RBS family

Via de novo synthesis

Since the RBS are short sequences, they can easily be ordered as two single-stranded complementary oligo's and annealed. See here for a tutorial on how to construct short parts via oligo annealing.

Via the Registry

The RBS can be obtained from the Registry distribution. These parts are cloned in plasmid pSB1A2, but there is also a constitutive promoter (derived from BBa_J23100) inserted into the XbaI site. So, for example, the EcoRI/PstI region of part J61100 reads:

...Biobrick Prefix..................J23100.................XbaI....RBS Part.....Biobrick Suffix...
gaattcgcggccgcttctagaGTTGACGGCTAGCTCAGTCCTAGGTACAGTGCTAGCTtctagaGAAAGAGGGGACAAactagtagcggccgctgcag

This feature in no way prevents the use of these parts in standard Biobrick assembly. Normal prefix insertion into EcoRI/XbaI will delete this promoter element. Suffix insertion into SpeI/PstI will retain this promoter, but it can of course be removed later by a prefix insertion.


Identifier Sequencea Strengthb
Master Sequence TCTAGAGAAAGANNNGANNNTACTAGATG
BBa_J61100 TCTAGAGAAAGAGGGGACAATACTAGATG
BBa_J61101 TCTAGAGAAAGACAGGACCCTACTAGATG
BBa_J61102 TCTAGAGAAAGATCCGATGTTACTAGATG
BBa_J61103 TCTAGAGAAAGATTAGACAATACTAGATG
BBa_J61104 TCTAGAGAAAGAAGGGACAGTACTAGATG
BBa_J61105 TCTAGAGAAAGAGGGGACAATACTAGATG
BBa_J61106 TCTAGAGAAAGAGGGGACAATACTAGATG
BBa_J61107 TCTAGAGAAAGAGGGGACAATACTAGATG
BBa_J61108 TCTAGAGAAAGAGGGGACAATACTAGATG
BBa_J61109 TCTAGAGAAAGAGGGGACAATACTAGATG
BBa_J61110 TCTAGAGAAAGAGGGGACAATACTAGATG
BBa_J61111 TCTAGAGAAAGAGGGGACAATACTAGATG
BBa_J61112 TCTAGAGAAAGAGGGGACAATACTAGATG
BBa_J61113 TCTAGAGAAAGAGGGGACAATACTAGATG
BBa_J61114 TCTAGAGAAAGAGGGGACAATACTAGATG
BBa_J61115 TCTAGAGAAAGAGGGGACAATACTAGATG
BBa_J61116 TCTAGAGAAAGAGGGGACAATACTAGATG
BBa_J61117 TCTAGAGAAAGAGGGGACAATACTAGATG
BBa_J61118 TCTAGAGAAAGAGGGGACAATACTAGATG
BBa_J61119 TCTAGAGAAAGAGGGGACAATACTAGATG
BBa_J61120 TCTAGAGAAAGAGGGGACAATACTAGATG
BBa_J61121 TCTAGAGAAAGAGGGGACAATACTAGATG
BBa_J61122 TCTAGAGAAAGAGGGGACAATACTAGATG
BBa_J61123 TCTAGAGAAAGAGGGGACAATACTAGATG
BBa_J61124 TCTAGAGAAAGAGGGGACAATACTAGATG
BBa_J61125 TCTAGAGAAAGAGGGGACAATACTAGATG
BBa_J61126 TCTAGAGAAAGAGGGGACAATACTAGATG
BBa_J61127 TCTAGAGAAAGAGGGGACAATACTAGATG
BBa_J61128 TCTAGAGAAAGAGGGGACAATACTAGATG
BBa_J61129 TCTAGAGAAAGAGGGGACAATACTAGATG
BBa_J61130 TCTAGAGAAAGAGGGGACAATACTAGATG
BBa_J61131 TCTAGAGAAAGAGGGGACAATACTAGATG
BBa_J61132 TCTAGAGAAAGAGGGGACAATACTAGATG
BBa_J61133 TCTAGAGAAAGAGGGGACAATACTAGATG
BBa_J61134 TCTAGAGAAAGAGGGGACAATACTAGATG
BBa_J61135 TCTAGAGAAAGAGGGGACAATACTAGATG
BBa_J61136 TCTAGAGAAAGAGGGGACAATACTAGATG
BBa_J61137 TCTAGAGAAAGAGGGGACAATACTAGATG
BBa_J61138 TCTAGAGAAAGAGGGGACAATACTAGATG
BBa_J61139 TCTAGAGAAAGAGGGGACAATACTAGATG

These parts are present in plasmid pSB1A2, but there is also a constitutive promoter (J23100-derived) inserted into the XbaI site. So, for example, the EcoRI/PstI region of part J61100 reads:

 Biobrick 5'    XbaI                    J23100              XbaI    RBS Part     Biobrick 3'
 gaattcgcggccgcttctagaGTTGACGGCTAGCTCAGTCCTAGGTACAGTGCTAGCTtctagaGAAAGAGGGGACAAactagtagcggccgctgcag

This feature in no way prevents the use of these parts in standard Biobrick assembly. Normal prefix insertion into EcoRI/XbaI will delete this promoter element. Suffix insertion into SpeI/PstI will retain this promoter, but it can of course be removed later by a prefix insertion.

Note also that the base 5' to the SpeI site is allowed to float in these parts and is therefore rarely "T". The "G" downstream of the XbaI site obeys the standard. Because the database does not permit variation at this position, the predicted sequences of composite parts derived from these parts will be incorrect at this position.

Characterization of the Ribosome Binding Site Library

042407pic1.jpg
To date, the RBS library has not been quantitatively characterized. This is largely a reflection of the method used to construct the library. Initiatially, all members of the library had the RBS variant upstream of an RFP gene. I measured red fluorescence for 384 individual library members, and the ranked activity of them is shown in the chart above. All screened clones showing activity detectable above background were combined into 7 activity pools. Each pool underwent a procedure designed to remove the RFP gene and put back the SpeI site in each part. 96 individual processed clones were then sequenced, and the family of RBS parts reflects all the unique clones.

So, at this stage we really can't say much about their relative activity. All I can tell you is that they should all initiate translation at a rate above background, and they are very roughly in decreasing order of activity. RBS's with low numbers are likely to be stronger RBS's than those with higher numbers.

Hopefully this will change in the near future, and if that happens the info. will be posted here.

...and of course YOU could do some characterization and post the info here.

The raw data for the library after sequenceing is Media:030807-Bca1050 raw rbs data.xls. The Tecan data of the clones before repooling / FokI fragmentation is Media:JCA_010607-TecanRBS.xls.

The original nomenclature was Bca1050 and then Bca9110.