Part:BBa_K2656008

Very Weak Ribosome Binding Site J61100

Figure 1. DNA basic parts domestication into GB. Second construction refers to RBS.

Part BBa_K2656008 is the original RBS BBa_J61100 standardized into the Golden Braid assembly method. Thus, it is a very weak 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 ribosome to bind to.

Complete Printeria RBS sequences collection is

Part	BioBrick	Description
BBa_K2656008	BBa_J61100	Very weak RBS
BBa_K2656009	BBa_B0030	Strong RBS
BBa_K2656010	BBa_B0032	Weak RBS
BBa_K2656011	BBa_B0034	Medium RBS
BBa_K2656012	BBa_J61101	Very weak RBS

Using the [http://2018.igem.org/Team:Valencia_UPV/Protocols Golden Gate assembly protocol], it can be combined with other Golden Braid 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 BsaI one-pot reaction.

Characterization of the this part was performed with the transcriptional unit BBa_K2656100, which was used in a comparative RBS expression experiment with composite parts BBa_K2656104 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_I746916 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 for each RBS.

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

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

Table 1. Optimized parameters for the BBa_K2656008 RBS.
Parameter	Value
Translation rate p	p = 0.04089 min-1
Dilution rate μ	μ = 0.01288 min-1

We have also calculated the relative force between the different RBS, taking BBa_K2656009 strong RBS as a reference. 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	0.042
p parameter ratio (pRBS/pref)	0.044

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]