Part:BBa_C0053

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

direction	Forward
function	Rep
ligands	?
protein	C2 P22
swisspro	P69202
tag	LVA

Functional Parameters: Austin_UTexas

Burden Imposed by this Part:

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 K_D	K_D (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:

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_D 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