Difference between revisions of "Part:BBa K1463600"

Latest revision as of 14:42, 1 November 2014

FliC

FliC encodes the major flagellar protein of E. coli and is thus fundamental to motility.

Figure 1 shows this biobrick used in conjunction with various promoters and ribosome binding sites to restore swimming in fliC knockout strains. This data is also shown as a histogram in Figure 2. These show that, with an appropriate promoter and RBS, this biobrick can restore swimming to almost wild-type levels.

We sequenced our fliC biobrick and found that it had the expected sequence, identical to fliC of the sequenced E. coli strain MG1655 at all positions except for the changes we had made to remove three PstI sites and one SpeI site. However, there were two coding differences between our biobrick and a previous fliC biobrick in the parts registry K777109. These differences are due to different source strains (We used MG1655 genomic DNA, they used E. coli strain K-12 substr. DH10B). It is unclear if these nonsynonymous changes alter flagella formation.

Flic Motility Swarm Assay

Figure 1: FliC Swarm Motility Assays.
(A) DS941, (B) DS941 ΔfliC,
(C) DS941 ΔfliC + pSB1C3 fliC (no promoter), (D) DS941 ΔfliC + J23100 (mutant promoter) fliC,
(E) DS941 ΔfliC + J23116-fliC(1), (F) DS941 ΔfliC + J23116-fliC(2),
(G) DS941 ΔfliC + J23106-fliC(1), (H) DS941 ΔfliC + J23106-fliC(2)

Flic Motility Histogram

Figure 2 - FliC Motility Histogram The promoters indicated in the histogram were used to drive the fliC biobrick K1463600 with the RBS B0034. Plasmids containing these constructs were used to complement a chromosomal fliC mutation. The diameter of swimming in a 16 hour swarm assay at 37 degrees is shown. The error bars indicate the range or results obtained in two repeats of the experiment. The strong J23100 promoter in this result contained a mutation rendering it inactive see K1463604.

For more information on the biobrick and methods used go to http://2014.igem.org/wiki/index.php?title=Team:Glasgow/Project/Mobility_Proteins#motA

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 1224
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 301
Illegal AgeI site found at 709
1000
COMPATIBLE WITH RFC[1000]

Protein data table for BioBrick BBa_K1463600 automatically created by the BioBrick-AutoAnnotator version 1.0

Nucleotide sequence in RFC 10: (underlined part encodes the protein)
ATGGCACAA ... CTCCAGGGTTAA
ORF from nucleotide position 1 to 1494 (excluding stop-codon)

Amino acid sequence: (RFC 25 scars in shown in bold, other sequence features underlined; both given below)

1
101
201
301
401

MAQVINTNSLSLITQNNINKNQSALSSSIERLSSGLRINSAKDDAAGQAIANRFTSNIKGLTQAARNANDGISVAQTTEGALSEINNNLQRVRELTVQAT
TGTNSESDLSSIQDEIKSRLDEIDRVSGQTQFNGVNVLAKNGSMKIQVGANDNQTITIDLKQIDAKTLGLDGFSVKNNDTVTTSAPVTAFGATTTNNIKL
TGITLSTEAATDTGGTNPASIEGVYTDNGNDYYAKITGGDNDGKYYAVTVANDGTVTMATGATANATVTDANTTKATTITSGGTPVQIDNTAGSATANLG
AVSLVKLQDSKGNDTDTYALKDTNGNLYAADVNETTGAVSVKTITYTDSSGAASSPTAVKLGGDDGKTEVVDIDGKTYDSADLNGGNLQTGLTAGGEALT
AVANGKTTDPLKALDDAIASVDKFRSSLGAVQNRLDSAVTNLNNTTTNLSEAQSRIQDADYATEVSNMSKAQIIQQAGNSVLAKANQVPQQVLSLLQG*

Sequence features: (with their position in the amino acid sequence, see the list of supported features)

None of the supported features appeared in the sequence

Amino acid composition:

Ala (A)	59 (11.8%)
Arg (R)	11 (2.2%)
Asn (N)	48 (9.6%)
Asp (D)	39 (7.8%)

Cys (C)	0 (0.0%)
Gln (Q)	27 (5.4%)
Glu (E)	14 (2.8%)
Gly (G)	44 (8.8%)

His (H)	0 (0.0%)
Ile (I)	28 (5.6%)
Leu (L)	37 (7.4%)
Lys (K)	25 (5.0%)

Met (M)	4 (0.8%)
Phe (F)	5 (1.0%)
Pro (P)	6 (1.2%)
Ser (S)	43 (8.6%)

Thr (T)	65 (13.1%)
Trp (W)	0 (0.0%)
Tyr (Y)	10 (2.0%)
Val (V)	33 (6.6%)

Amino acid counting

	Total number:	498
	Positively charged (Arg+Lys):	36 (7.2%)
	Negatively charged (Asp+Glu):	53 (10.6%)
	Aromatic (Phe+His+Try+Tyr):	15 (3.0%)

Biochemical parameters

	Atomic composition:	C₂₁₆₃H₃₅₅₃N₆₃₁O₇₉₈S₄
	Molecular mass [Da]:	51295.0
	Theoretical pI:	4.50
	Extinction coefficient at 280 nm [M^-1 cm^-1]:	14900 / 14900 (all Cys red/ox)

Plot for hydrophobicity, charge, predicted secondary structure, solvent accessability, transmembrane helices and disulfid bridges

Codon usage

	Organism:	E. coli	B. subtilis	S. cerevisiae	A. thaliana	P. patens	Mammals
	Codon quality (CAI):	excellent (0.83)	good (0.78)	good (0.68)	good (0.74)	excellent (0.83)	good (0.70)

Alignments (obtained from PredictProtein.org)

	SwissProt:	P04949 (100% identity on 498 AAs), P06179 (47% identity on 495 AAs)
	TrEML:	L2Z8U7 (100% identity on 498 AAs), M2PKZ4 (100% identity on 498 AAs), C6EBD8 (100% identity on 498 AAs), C9QSQ0 (100% identity on 498 AAs), F9QUT2 (100% identity on 498 AAs), G2EY21 (100% identity on 498 AAs), I0ZSG6 (100% identity on 498 AAs), C4ZQK1 (100% identity on 498 AAs), Q53ZW4 (100% identity on 498 AAs), U6N709 (100% identity on 498 AAs)
	PDB:	-

Predictions (obtained from PredictProtein.org)

Subcellular Localization (reliability in brackets)

	Archaea:	cytosol (100%)
	Bacteria:	secreted (65%)
	Eukarya:	nucleus (55%)

Gene Ontology (reliability in brackets)

	Molecular Function Ontology:	GO:0005515 (100%)
	Biological Process Ontology:	GO:0009405 (9%), GO:0001539 (63%)

Predicted features:

	Disulfid bridges:	-
	Transmembrane helices:	-

The BioBrick-AutoAnnotator was created by TU-Munich 2013 iGEM team. For more information please see the documentation.
If you have any questions, comments or suggestions, please leave us a comment.

@@ Line 6: / Line 6: @@
 <br>Figure 1 shows this biobrick used in conjunction with various promoters and ribosome binding sites to restore swimming in fliC knockout strains. This data is also shown as a histogram in Figure 2. These show that, with an appropriate promoter and RBS, this biobrick can restore swimming to almost wild-type levels.
-<br> We sequenced our fliC biobrick and found that it had the expected sequence, identical to fliC of the sequenced E. coli strain MG1655 at all positions except for the changes we had made to remove three PstI sites and one SpeI site. However, there were two coding differences between our biobrick and a previous fliC biobrick in the parts registry [http://www.https://parts.igem.org/Part:BBa_K777109 K777109]. K777109 has mutations G145T and T331G in the DNA resulting in amino acid changes A49S and S111A. These two changes appear to be mutations in K777109 and thus our biobrick (BBa_K1463600) is an improved version of K777109.
+<br> We sequenced our fliC biobrick and found that it had the expected sequence, identical to fliC of the sequenced E. coli strain MG1655 at all positions except for the changes we had made to remove three PstI sites and one SpeI site. However, there were two coding differences between our biobrick and a previous fliC biobrick in the parts registry [https://parts.igem.org/Part:BBa_K777109 K777109]. These differences are due to different source strains (We used MG1655 genomic DNA, they used E. coli strain K-12 substr. DH10B). It is unclear if these nonsynonymous changes alter flagella formation.
 <br>
 ==Flic Motility Swarm Assay==
@@ Line 17: / Line 17: @@
 ==Flic Motility Histogram==
 https://static.igem.org/mediawiki/2014/f/fc/GU_Figure_2_Motility_histogram.png
-<br><b>Figure 2 - FliC Motility Histogram</b>
+<br><b>Figure 2 - FliC Motility Histogram</b> The promoters indicated in the histogram were used to drive the fliC biobrick K1463600 with the RBS B0034. Plasmids containing these constructs were used to complement a chromosomal fliC mutation. The diameter of swimming in a 16 hour swarm assay at 37 degrees is shown. The error bars indicate the range or results obtained in two repeats of the experiment. The strong J23100 promoter in this result contained a mutation rendering it inactive [https://parts.igem.org/Part:BBa_K1463604 see K1463604].
 <br><br>