Part:BBa_K2656012

Very Low Ribosome Binding Site J61101

Figure 1. DNA basic parts domestication. Second figure refers to RBS basic parts.

BBa_K2656012 is the J61101 ribosome binding site standardized into the Golden Braid assembly method. Thus, it is strong RBS 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 [http://2018.igem.org/Team:Valencia_UPV/Part_Collection 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_K2656104, which was used in a comparative RBS expression experiment with composite parts BBa_K265610 and BBa_K2656101. They all were assembled in a Golden Braid alpha1 plasmid using the same promoter, CDS and terminator:

• Promoter BBa_K2656004: the J23106 promoter in its Golden Braid compatible version from our [http://2018.igem.org/Team:Valencia_UPV/Part_Collection Part Collection]
• CDS BBa_K2656013: the BBa_K2656009 sfGFP sequence in its Golden Braid standardized version from our [http://2018.igem.org/Team:Valencia_UPV/Part_Collection Part Collection]
• Terminator BBa_K2656026: the B0015 transcriptional terminator in its Golden Braid compatible version from our [http://2018.igem.org/Team:Valencia_UPV/Part_Collection Part Collection]

By using this [http://2018.igem.org/Team:Valencia_UPV/Experiments#exp_protocol experimental protocol], we have obtained the parameters to valide our [http://2018.igem.org/Team:Valencia_UPV/Modeling#models constitutive model]and rationale choose its optimized values based on each RBS.

Figure 1. RBS expression experiment with K2656008, K2656009 and K2656012 RBS basic pieces

Figure 2. RBS expression experiment with K2656008 and K2656012 RBS basic pieces

Table 1. Optimized parameters for the BBa_K2656008 RBS.
Parameter	Value
Translation rate p	p = 0.02889 min-1
Dilution rate μ	μ = 0.0118 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 relative strength and p ratio.
Parameter	Value
Relative strength	0.032
p parameter ratio (pRBS/pref)	0.032

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]