Difference between revisions of "Part:BBa K2656008"
 
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K2656008 short</partinfo>
 
<partinfo>BBa_K2656008 short</partinfo>
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[[File:T--Valencia_UPV--im11UPV2018.png|250px|thumb|centro|alt=domestication.|Figure 1. DNA basic parts domestication into GB. Second construction refers to RBS.]]
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Part BBa_K2656008 is the original RBS [https://parts.igem.org/Part:BBa_J61100 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.
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This RBS is part of our complete '''Printeria RBS Basic Part Collection'''
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{| class="wikitable" style="text-align: center; color: black;"
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|Part
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|BioBrick
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|Description
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|Strength (Relative to BBa_K2656009)
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|-
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|[https://parts.igem.org/Part:BBa_K2656009 BBa_K2656009]
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|[https://parts.igem.org/Part:BBa_B0030 BBa_B0030]
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|Strong RBS
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|1
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|-
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|[https://parts.igem.org/Part:BBa_K2656011 BBa_K2656011]
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|[https://parts.igem.org/Part:BBa_B0034 BBa_B0034]
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|Medium RBS
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|0.38
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|-
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|[https://parts.igem.org/Part:BBa_K2656010 BBa_K2656010]
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|[https://parts.igem.org/Part:BBa_B0032 BBa_B0032]
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|Weak RBS
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|0.047
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|-
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|[https://parts.igem.org/Part:BBa_K2656008 BBa_K2656008]
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|[https://parts.igem.org/Part:BBa_J61100 BBa_J61100]
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|Very weak RBS
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|0.042
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|-
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|[https://parts.igem.org/Part:BBa_K2656012 BBa_K2656012]
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|[https://parts.igem.org/Part:BBa_J61101 BBa_J61101]
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|Very weak RBS
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|0.032
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|-
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|}
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Using the <html><a href="https://static.igem.org/mediawiki/parts/b/bc/T--Valencia_UPV--protocols.pdf#page=27"> Golden Gate assembly protocol </a></html>, 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.
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Characterization of this part was performed with the transcriptional unit [https://parts.igem.org/Part:BBa_K2656100 BBa_K2656100], which was used in a comparative RBS expression experiment with composite parts [https://parts.igem.org/Part:BBa_K2656104 BBa_K2656104] and [https://parts.igem.org/Part:BBa_K2656101 BBa_K2656101].
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They all were assembled in a Golden Braid alpha1 plasmid using the same promoter, CDS and terminator:
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<html>
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<ul>
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<li></html>Promoter [https://parts.igem.org/Part:BBa_K2656004 BBa_K2656004]: the [https://parts.igem.org/Part:BBa_J23106 J23106] promoter in its Golden Braid compatible version from our [http://2018.igem.org/Team:Valencia_UPV/Part_Collection Part Collection]<html></li>
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<li></html>CDS [https://parts.igem.org/Part:BBa_K2656013 BBa_K2656013]: the [https://parts.igem.org/Part:BBa_I746916 BBa_I746916] sfGFP sequence in its Golden Braid standardized version from our [http://2018.igem.org/Team:Valencia_UPV/Part_Collection Part Collection]<html></li>
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<li></html>Terminator [https://parts.igem.org/Part:BBa_K2656026 BBa_K2656026]: the [https://parts.igem.org/Part:BBa_B0015 B0015] transcriptional terminator in its Golden Braid compatible version from our [http://2018.igem.org/Team:Valencia_UPV/Part_Collection Part Collection]<html></li>
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</ul>
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</html>
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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.
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[[File:T--Valencia_UPV--optimization_exp2_RBS_all_graph_expUPV2018.png|900px|thumb|none|alt=RBS experiment.|Figure 1. RBS expression experiment with K2656008, K2656009 and K2656012 basic pieces]]
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[[File:T--Valencia_UPV--optimization_exp2_RBS_2obj_graph_expUPV2018.png|900px|thumb|none|alt=RBS experiment.|Figure 2. RBS expression experiment with K2656008 and K2656012 basic pieces]]
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{|class='wikitable'
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|colspan=4|Table 1. Optimized parameters for the BBa_K2656008 RBS.
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|-
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|'''Parameter'''
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|'''Value'''
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|-
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|Translation rate p
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|p = 0.04089 min-1
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|-
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|Dilution rate μ
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|μ = 0.01288 min-1
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|}
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We have also calculated the relative force between the different RBS, taking [https://parts.igem.org/Part:BBa_K2656009 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 the reference RBS. Likewise, a ratio between p parameters of the different RBS parts and p parameter of the reference RBS has been calculated.
  
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Part BBa_J61100  domesticated to the Golden Braid 3.0 grammar. It can be used to assembly transcriptional units with the Golden Gate assembly method, and also, it can be used as a Biobrick.
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{|class='wikitable'
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|colspan=4|Table 2. BBa_K2656008 relative strength and p ratio.
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|-
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|'''Parameter'''
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|'''Value'''
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|-
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|Relative strength
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|0.042
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|-
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|p parameter ratio (pRBS/pref)
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|0.044
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|}
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 00:00, 18 October 2018

Very Weak Ribosome Binding Site J61100

domestication.
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.

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 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 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.

RBS experiment.
Figure 1. RBS expression experiment with K2656008, K2656009 and K2656012 basic pieces
RBS experiment.
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. 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 the 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 0.042
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]