RBS

Part:BBa_K2656009

Designed by: Adrián Requena Gutiérrez, Carolina Ropero   Group: iGEM18_Valencia_UPV   (2018-09-20)

Strong Ribosome Binding Site B0030

domestication.
Figure 1. DNA basic parts domestication

BBa_K2656009 is the BBa_B0030 RBS standardized into the Golden Braid assembly method. Thus, it is strong ribosome binding site compatible both with the BioBrick and Golden Gate grammar. It also includes the BioBrick equivalent scar in the 3' extreme, so the insertion of this supplementary bases ensure correct spacing for the CDS expression when assembled into a TU.


This RBS is part of our complete Printeria RBS Basic Part Collection

Part BioBrick Description Strength (Relative to BBa_K2656009)
BBa_K2656009 BBa_B0030 Strong RBS 1
BBa_K2656011 BBa_B0034 Medium RBS 0.38
BBa_K2656010 BBa_B0032 Weak RBS 0.047
BBa_K2656008 BBa_J61100 Very weak RBS 0.042
BBa_K2656012 BBa_J61101 Very weak RBS 0.032


Using the Golden Gate assembly protocol , it can be combined with other GB adapted basic parts, such as the ones from our Valencia UPV IGEM 2018 Printeria Collection, to assemble transcriptional units in a single one-pot reaction.


Characterization of this part was performed with the transcriptional unit BBa_K2656101, which was used in two comparative RBS expression experiments with composite parts BBa_K2656102, BBa_K2656103 and BBa_K2656100, BBa_K2656104 They all were assembled in a Golden Braid alpha1 plasmid using the same promoter, CDS and terminator:

By using this experimental protocol, we have obtained the parameters to valide our constitutive modeland rationale choose its optimized values based on each RBS tested.

RBS experiment 1.
Figure 2. RBS expression experiment with K2656009, K26560010 and K2656011 RBS basic parts
RBS experiment 2.
Figure 3. RBS expression experiment with K2656009, K2656008 and K2656012 RBS basic parts


Table 1. Optimized parameters for the BBa_K2656009 RBS Experiment 1.
Parameter Value
Translation rate p p = 0.2059 min-1
Dilution rate μ μ = 0.01492 min-1
Table 1. Optimized parameters for the BBa_K2656009 RBS Experiment 2.
Parameter Value
Translation rate p p = 0.9256 min-1
Dilution rate μ μ = 0.01197 min-1

We have also calculated the relative force between the different RBS, taking BBa_K2656009 strong RBS as a reference. It has been defined as the quotient between the values of the protein in equilibrium of the results of the simulation of one RBS and another reference RBS. Likewise, a ratio between p parameters of the different RBS parts and p parameter of the reference RBS has been calculated.

Table 2. BBa_K2656008 (GB BBa_B0032 RBS) relative strength and p ratio.
Parameter Value
Relative strength 1
p parameter ratio (pRBS/pref) 1


Sequence and Features

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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Categories
//rbs/prokaryote/constitutive/community
//ribosome/prokaryote/ecoli
//chassis/prokaryote/ecoli
//direction/forward
//regulation/constitutive
Parameters
biology