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Revision as of 03:12, 20 October 2016
The Circuit Control Toolbox
The Circuit Control Toolbox consists of a diverse collection of biological parts that provide precise control over the behavior of any arbitrary genetic circuit. It encompasses a series of components able to achieve a range of modifications; additionally, we provide tools that may be used to measure and characterize the parts within the toolbox itself. The Toolbox contains a total of 118 unique Biobrick parts, many of which have been sequence-confirmed and characterized on multiple BioBrick backbones.
Every part submitted in our Toolbox is flanked by standard Unique Nucleotide Sequence (UNS) segments. These segments, generated by Pam Silver and colleagues at Harvard University, were designed to facilitate insulated assembly of complex and repeat-heavy components without biological interference. They consist of randomized sequences of nucleotides which have been optimized for low hairpin and heterodimer formation frequency; they were further checked to ensure minimal overlap with promoter-like sequences, start codons, and the E. coli genome sequence. By flanking part inserts with distinct UNS sequences, one can achieve secure and efficient synthesis of multiple-gene networks [1].
We chose to standardize each part in our Toolbox with an upstream UNS2 and downstream UNS3 sequence. These sequences were chosen based on their compatibility with standard BioBrick enzymes, as well as the BsmBI enzyme used for Iterative Capped Assembly; UNS1 was thus eliminated from consideration, as it contained the recognition site for BsmBI. By surrounding each part by these two UNS sequences, we were able to make all of our parts far more compatible with Gibson Assembly than the standard Biobrick Prefix/Suffix regions. UNS-guided Gibson Assembly, using UNS regions directly interior to the Biobrick Prefix and Suffix, provides the advantages of significantly decreased homodimer affinity compared to Gibson Assembly on the Prefix/Suffix regions, while maintaining the 3A assembly capability of the Biobrick standard.
Many of the parts in the Circuit Control Toolbox can be categorized into one of five subsections based on their function. These include the following:
RBS Characterization Constructs These parts are IPTG-inducible constructs which contain self-cleaving ribozyme RiboJ immediately upstream of the RBS sequence, which insulates the contribution of the RBS to gene expression from contributions from the 5’ UTR region. The inclusion of this element provides an unprecedented avenue for standardizing RBS measurements regardless of associated coding sequences.
Promoter Characterization Constructs This category consists of the Anderson Library of promoters driving our B0034 RBS Characterization construct; each promoter construct was further characterized with and without RiboJ insertion immediately upstream of the RBS. The purpose of these constructs is to investigate and characterize the impact of RiboJ on absolute gene expression level.
Decoy Binding Array Construction Kit Here we provide an 85-repeat array of tetO binding sites on the BioBrick backbone. Furthermore, we offer a suite of parts which can be used to construct arrays of arbitrary length using Iterative Capped Assembly. These parts are doubly-flanked by UNS regions (UNS 2, 4, 5, and 3) to facilitate UNS-guided Iterative Capped Assembly [link to protocol page], which can be used to construct arrays which are compatible with the UNS standard.
Synthetic Enhancer Suite This is a collection of UNS-flanked BioBrick standard parts associated with the Synthetic Enhancer project. In addition to our well-characterized 3x tetO Synthetic Enhancer part (BBa_K2066114), we provide additional variants in which we express sfGFP rather than mCherry for stronger readout; furthermore, we include a constitutive tetR expression cassette on the same part so that the synthetic enhancer component can be characterized completely using a single plasmid. In order to facilitate the construction of additional variants of the Synthetic Enhancer, we include intermediate construction parts, such as the functional NRII coding region, on UNS-flanked BioBrick backbone.
UNS-Standardized Repressor/Reporter Constructs
This collection of parts provides a useful avenue for testing other parts within the Circuit Control Toolbox; this is accomplished by simulating arbitrary circuit input and being able to visualize the repressor concentration. The collection includes tetR-mCherry and lacI-mCherry fusions under the pBad promoter taken from Daniel et al [2].
1. Torella, J. P., Boehm, C. R., Lienert, F., Chen, J., Way, J. C., & Silver, P. A. (2014). Rapid construction of insulated genetic circuits via synthetic sequence-guided isothermal assembly. Nucleic Acids Research,42(1), 681-689.doi:10.1093/nar/gkt860
2. Daniel, R., Rubens, J. R., Sarpeshkar, R., & Lu, T. K. (2013). Synthetic analog computation in living cells.Nature, 497, 619-623.
doi:10.1038/nature12148
Part Collection
Part
Description
K2066034
Promoter and RBS characterization construct – mCherry + Spinach
K2066035
plLac0-1 RBS Characterization Part – B0034
K2066036
plLac0-1 RBS Characterization Part – B0031
K2066044
plLac0-1 RBS Characterization Part – B0029
K2066045
plLac0-1 RBS Characterization Part – B0064
K2066046
plLac0-1 RBS Characterization Part – B0030
K2066047
plLac0-1 RBS Characterization Part – B0035
K2066048
plLac0-1 RBS Characterization Part – B0032
K2066049
plLac0-1 RBS Characterization Part – B0033
Part
Description
K2066034
Promoter and RBS characterization construct – mCherry + Spinach
K2066035
plLac0-1 RBS Characterization Part – B0034
K2066040
J23100 + RiboJ Promoter Characterization Part with 2x Broccoli
K2066041
J23100 without RiboJ Promoter Characterization Part
K2066042
J23115 + RiboJ Promoter Characterization Part
K2066043
J23115 without RiboJ Promoter Characterization Part
K2066050
F30 scaffold/2x Broccoli
K2066051
J23107 with RiboJ Promoter Characterization Part
K2066052
J23107 without RiboJ Promoter Characterization Part
K2066054
T7 Promoter with RiboJ Promoter Characterization Part
K2066055
T7 Promoter without RiboJ Promoter Characterization Part
K2066057
Promoter characterization part – strong RBS-sfGFP with J23100
K2066058
Promoter characterization part – strong RBS-sfGFP with J23115
K2066059
Promoter characterization part – B0034-sfGFP with J23100, without RiboJ
K2066060
Promoter characterization part – B0034-sfGFP with J23115, without RiboJ
K2066061
Promoter characterization part – RiboJ-B0034-sfGFP with J23101
K2066062
Promoter characterization part – RiboJ-B0034-sfGFP with J23102
K2066063
Promoter characterization part – RiboJ-B0034-sfGFP with J23103
K2066064
Promoter characterization part – RiboJ-B0034-sfGFP with J23104
K2066065
Promoter characterization part – RiboJ-B0034-sfGFP with J23105
K2066066
Promoter characterization part – RiboJ-B0034-sfGFP with J23106
K2066067
Promoter characterization part – RiboJ-B0034-sfGFP with J23107
K2066068
Promoter characterization part – RiboJ-B0034-sfGFP with J23108
K2066069
Promoter characterization part – RiboJ-B0034-sfGFP with J23109
K2066070
Promoter characterization part – RiboJ-B0034-sfGFP with J23110
K2066071
Promoter characterization part – RiboJ-B0034-sfGFP with J23111
K2066072
Promoter characterization part – RiboJ-B0034-sfGFP with J23112
K2066073
Promoter characterization part – RiboJ-B0034-sfGFP with J23113
K2066074
Promoter characterization part – RiboJ-B0034-sfGFP with J23114
K2066075
Promoter characterization part – RiboJ-B0034-sfGFP with J23116
K2066076
Promoter characterization part – RiboJ-B0034-sfGFP with J23117
K2066077
Promoter characterization part – RiboJ-B0034-sfGFP with J23118
K2066078
Promoter characterization part – RiboJ-B0034-sfGFP with J23119
K2066079
Promoter characterization part – RiboJ-B0034-sfGFP with R0040
K2066080
Promoter characterization part – RiboJ-B0034-sfGFP with R0010
K2066081
Promoter characterization part – RiboJ-B0034-sfGFP with J23150
K2066082
Promoter characterization part – RiboJ-B0034-sfGFP with J23151
K2066083
Promoter characterization part – B0034-sfGFP with J23101
K2066084
Promoter characterization part – B0034-sfGFP with J23102
K2066085
Promoter characterization part – B0034-sfGFP with J23103
K2066086
Promoter characterization part – B0034-sfGFP with J23104
K2066087
Promoter characterization part – B0034-sfGFP with J23105
K2066088
Promoter characterization part – B0034-sfGFP with J23106
K2066089
Promoter characterization part – B0034-sfGFP with J23107
K2066090
Promoter characterization part – B0034-sfGFP with J23108
K2066091
Promoter characterization part – B0034-sfGFP with J23109
K2066092
Promoter characterization part – B0034-sfGFP with J23110
K2066093
Promoter characterization part – B0034-sfGFP with J23111
K2066094
Promoter characterization part – B0034-sfGFP with J23112
K2066095
Promoter characterization part – B0034-sfGFP with J23113
K2066096
Promoter characterization part – B0034-sfGFP with J23114
K2066097
Promoter characterization part – B0034-sfGFP with J23116
K2066098
Promoter characterization part – B0034-sfGFP with J23117
K2066099
Promoter characterization part – B0034-sfGFP with J23118
K20660100
Promoter characterization part – B0034-sfGFP with J23119
K20660101
Promoter characterization part – B0034-sfGFP with R0040
K20660102
Promoter characterization part – RiboJ-B0034-sfGFP with R0011
K20660103
K20660104
Promoter characterization part – B0034-sfGFP with R0010 without RiboJ
K20660105
Promoter characterization part – B0034-sfGFP with R0011 without RiboJ
K20660106
Promoter characterization part – B0034-sfGFP with J23150 without RiboJ
K20660107
Promoter characterization part – B0034-sfGFP with J23151 without RiboJ
K2066108
Promoter characterization part – RiboJ-B0034-sfGFP with J23107
K2066048
Promoter characterization part – B0034-sfGFP with J23107 without RiboJ
K2066049
Promoter characterization part – RiboJ-B0034-sfGFP with J23100
K2066049
Promoter characterization part – RiboJ-B0034-sfGFP with J23115
Part
Description
K2066034
Promoter and RBS characterization construct – mCherry + Spinach
plLac0-1 RBS Characterization Part
K2066035
– B0034
K2066036
plLac0-1 RBS Characterization Part – B0031
K2066044
plLac0-1 RBS Characterization Part – B0029
K2066045
plLac0-1 RBS Characterization Part – B0064
K2066046
plLac0-1 RBS Characterization Part – B0030
K2066047
plLac0-1 RBS Characterization Part – B0035
K2066048
plLac0-1 RBS Characterization Part – B0032
K2066049
plLac0-1 RBS Characterization Part – B0033
Part
Description
K2066034
Promoter and RBS characterization construct – mCherry + Spinach
K2066035
plLac0-1 RBS Characterization Part – B0034
K2066036
plLac0-1 RBS Characterization Part – B0031
K2066044
plLac0-1 RBS Characterization Part – B0029
K2066045
plLac0-1 RBS Characterization Part – B0064
K2066046
plLac0-1 RBS Characterization Part – B0030
K2066047
plLac0-1 RBS Characterization Part – B0035
K2066048
plLac0-1 RBS Characterization Part – B0032
K2066049
plLac0-1 RBS Characterization Part – B0033
Part
Description
K2066034
Promoter and RBS characterization construct – mCherry + Spinach
K2066035
plLac0-1 RBS Characterization Part – B0034
K2066036
plLac0-1 RBS Characterization Part – B0031
K2066044
plLac0-1 RBS Characterization Part – B0029
K2066045
plLac0-1 RBS Characterization Part – B0064
K2066046
plLac0-1 RBS Characterization Part – B0030
K2066047
plLac0-1 RBS Characterization Part – B0035
K2066048
plLac0-1 RBS Characterization Part – B0032
K2066049
plLac0-1 RBS Characterization Part – B0033
References