Difference between revisions of "Part:BBa C0053"
 
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<b>Summary:</b>
 
<b>Summary:</b>
 
</li>
 
</li>
 +
P22 c2 repressor DNA binding site.
 +
 +
{| class=wikitable
 +
|-
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!Part Name
 +
!Part number
 +
!Sequence
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!Rel K<sub>D</sub>
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!K<sub>D</sub> (in M)
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|-
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|P22 binding site A
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|<partinfo>BBa_K3889080</partinfo>
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|ATTTAAGATATCTTAAAT
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|1
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|1.6 × 10<sup>-8</sup>
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|-
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|P22 binding site B
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|<partinfo>BBa_K3889081</partinfo>
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|AATTAAGATATCTTAATT
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|1.8
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|2.88 × 10<sup>-8</sup>
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|-
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|P22 binding site C
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|<partinfo>BBa_K3889082</partinfo>
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|ATTTAAGAATTCTTAAAT
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|2
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|3.2 × 10<sup>-8</sup>
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|-
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|P22 binding site D
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|<partinfo>BBa_K3889083</partinfo>
 +
|AGTTAAGATATCTTAACT
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|2.6
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|4.16 × 10<sup>-8</sup>
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|-
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|P22 binding site E
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|<partinfo>BBa_K3889084</partinfo>
 +
|ATTAAAGATATCTTTAAT
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|3.8
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|6.08 × 10<sup>-8</sup>
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|-
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|P22 binding site F
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|<partinfo>BBa_K3889085</partinfo>
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|ACTTAAGATATCTTAAGT
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|4.3
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|6.88 × 10<sup>-8</sup>
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|-
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|P22 binding site G
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|<partinfo>BBa_K3889086</partinfo>
 +
|ATTCAAGATATCTTGAAT
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|5
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|8.0 × 10<sup>-8</sup>
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|-
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|P22 binding site H
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|<partinfo>BBa_K3889087</partinfo>
 +
|ATTGAAGATATCTTCAAT
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|7.6
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|1.216 × 10<sup>-7</sup>
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|-
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|P22 binding site I
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|<partinfo>BBa_K3889088</partinfo>
 +
|ATTTAAGAGCTCTTAAAT
 +
|10
 +
|1.6 × 10<sup>-7</sup>
 +
|-
 +
|P22 binding site J
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|<partinfo>BBa_K3889089</partinfo>
 +
|ATTTAAGACGTCTTAAAT
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|10
 +
|1.6 × 10<sup>-7</sup>
 +
|-
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|P22 binding site K
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|<partinfo>BBa_K3889090</partinfo>
 +
|ATTTACGATATCGTAAAT
 +
|30
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|4.8 × 10<sup>-7</sup>
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|-
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|P22 binding site L
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|<partinfo>BBa_K3889091</partinfo>
 +
|ATTTAAAATATTTTAAAT
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|55
 +
|8.8 × 10<sup>-7</sup>
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|}
 +
 +
 +
[[File:T--IISER-Tirupati_India--Kd_values_of_P22_binding_site.png|thumb|center|400px|Fig.1:Kd values of P22 binding site]]<br>
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 +
The alignment of these sequences are as follows:
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 +
[[File:T--IISER-Tirupati India--P22 binding sites allignment.jpg]]<br>
 +
 +
Fig source: Geneious version 2021.2 created by Biomatters. Available from https://www.geneious.com
 +
 +
===Usage and Biology===
 +
Different binding affinities of a repressor provides a variable system that can be used for different types of systems. As K<sub>D</sub> increases the dissociation of repressor from the DNA sequence also increases providing with lower repression. P22 repressor binds to this sequence as a dimer. This inhibits the enzymes from transcription the genes on whose promoter this operator site is present. Hence this could be fused with any promoter in order to form a repressible system as done in <partinfo>BBa_K3889102</partinfo>.
 +
 +
 +
 +
===Another Contribution: Modification in sequence===
 
<partinfo>BBa_K3889020</partinfo> is a modified version specific to our project's assembly.
 
<partinfo>BBa_K3889020</partinfo> is a modified version specific to our project's assembly.
 
The following modifications were made:
 
The following modifications were made:
Line 59: Line 157:
 
*LVA tag Removed
 
*LVA tag Removed
 
*barcodes Removed
 
*barcodes Removed
 +
 +
===References===
 +
1. Watkins, D., Hsiao, C., Woods, K. K., Koudelka, G. B., & Williams, L. D. (2008). P22 c2 Repressor−Operator Complex:  Mechanisms of Direct and Indirect Readout. Biochemistry, 47(8), 2325–2338. https://doi.org/10.1021/bi701826f

Latest revision as of 23:33, 21 October 2021

c2 repressor from Salmonella phage P22 (+LVA)

The P22 c2 repressor protein coding sequence is a 720 base-pair sequence with the standard RBS-compatible BioBrick prefix and the standard BioBrick suffix sections on its ends. It binds to the P22 c2 regulatory sequence, BBa_R0053. The sequence contains a LVA tag for faster degradation.


Usage and Biology

P22 c2 is a member of the lamboid cI protein family.

The coding sequence has been modified from wild type c2 to contain an LVA tag,

and two TAA stop codons.


Sequence and Features


Barcodes are discontinued, but one was appended to the sequence of this part. Composite parts using this part will include the barcode. More ...

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
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 166


Functional Parameters

directionForward
functionRep
ligands?
proteinC2 P22
swissproP69202
tagLVA



Functional Parameters: Austin_UTexas

Burden Imposed by this Part:

Burden Value: 27.1 ± 6.5%

Burden is the percent reduction in the growth rate of E. coli cells transformed with a plasmid containing this BioBrick (± values are 95% confidence limits). This part exhibited a significant burden. Users should be aware that BioBricks with a burden of >20-30% may be susceptible to mutating to become less functional or nonfunctional as an evolutionary consequence of this fitness cost. This risk increases as they used for more bacterial cell divisions or in larger cultures. Users should be especially careful when combining multiple burdensome parts, as plasmids with a total burden of >40% are expected to mutate so quickly that they become unclonable. Refer to any one of the BBa_K3174002 - BBa_K3174007 pages for more information on the methods and other conclusions from a large-scale measurement project conducted by the 2019 Austin_UTexas team.

This functional parameter was added by the 2020 Austin_UTexas team.


Modification in Sequence : iGEM21_IISER-Tirupati_India

  • Group: iGEM21_IISER-Tirupati_India
  • Author: Prameya Garge, Ashwin Sharma
  • Summary:

    • P22 c2 repressor DNA binding site.

    Part Name Part number Sequence Rel KD KD (in M)
    P22 binding site A BBa_K3889080 ATTTAAGATATCTTAAAT 1 1.6 × 10-8
    P22 binding site B BBa_K3889081 AATTAAGATATCTTAATT 1.8 2.88 × 10-8
    P22 binding site C BBa_K3889082 ATTTAAGAATTCTTAAAT 2 3.2 × 10-8
    P22 binding site D BBa_K3889083 AGTTAAGATATCTTAACT 2.6 4.16 × 10-8
    P22 binding site E BBa_K3889084 ATTAAAGATATCTTTAAT 3.8 6.08 × 10-8
    P22 binding site F BBa_K3889085 ACTTAAGATATCTTAAGT 4.3 6.88 × 10-8
    P22 binding site G BBa_K3889086 ATTCAAGATATCTTGAAT 5 8.0 × 10-8
    P22 binding site H BBa_K3889087 ATTGAAGATATCTTCAAT 7.6 1.216 × 10-7
    P22 binding site I BBa_K3889088 ATTTAAGAGCTCTTAAAT 10 1.6 × 10-7
    P22 binding site J BBa_K3889089 ATTTAAGACGTCTTAAAT 10 1.6 × 10-7
    P22 binding site K BBa_K3889090 ATTTACGATATCGTAAAT 30 4.8 × 10-7
    P22 binding site L BBa_K3889091 ATTTAAAATATTTTAAAT 55 8.8 × 10-7


    Fig.1:Kd values of P22 binding site

    The alignment of these sequences are as follows:

    T--IISER-Tirupati India--P22 binding sites allignment.jpg

    Fig source: Geneious version 2021.2 created by Biomatters. Available from https://www.geneious.com

    Usage and Biology

    Different binding affinities of a repressor provides a variable system that can be used for different types of systems. As KD increases the dissociation of repressor from the DNA sequence also increases providing with lower repression. P22 repressor binds to this sequence as a dimer. This inhibits the enzymes from transcription the genes on whose promoter this operator site is present. Hence this could be fused with any promoter in order to form a repressible system as done in BBa_K3889102.


    Another Contribution: Modification in sequence

    BBa_K3889020 is a modified version specific to our project's assembly. The following modifications were made:

    • Spa1 site Removed
    • LVA tag Removed
    • barcodes Removed

    References

    1. Watkins, D., Hsiao, C., Woods, K. K., Koudelka, G. B., & Williams, L. D. (2008). P22 c2 Repressor−Operator Complex:  Mechanisms of Direct and Indirect Readout. Biochemistry, 47(8), 2325–2338. https://doi.org/10.1021/bi701826f