Coding
motB

Part:BBa_K1463750

Designed by: Beth Greig   Group: iGEM14_Glasgow   (2014-10-04)

MotB

MotB is a motility gene encoding a membrane protein which is a part of the stator of the flagellar motor (Fig. 1).

This biobrick is an improvement of BBa_K777117[1]. Changes made can be found in the design section.


F1.medium.gif

Fig. 1: Flagellar motor scheme. Picture taken from Reid et al., 2006[2].


Usage and Biology

MotA and motB are expressed from a flagellar motor gene operon, and both of these genes are required for motor function, hence swimming. Deletions in upstream genes in operons are often known to have “polar” effects, disrupting expression of downstream genes. Therefore in our DS941 ΔmotA strain deletion is believed to be reducing expression of motB. To test this, we made a motA-motB biobrick and check whether it restores swimming to our delta-motA mutant. We inserted the BBa_B0032 RBS – motA - motB biobrick into the BBa_J61002 vector containing a variety of different promoters from the parts distribution: BBa_J23106 (½ the strength of J23100) BBa_J23116 (¼ the strength of J23100) BBa_J23103 (very weak promoter) BBa_J23112 (weakest promoter we could find in the registry, barely any expression) (Strength measured with RFP: Part BBa_J23100)

We then used swarm assays (semi-solid agar motility test) to investigate whether these plasmids would rescue swimming of a motA mutant. The results of the swarm assays are shown in Figure 1. While none of the plasmids containing only motA restored swimming to the mutant to any significant extent, with motA-motB we saw a significantly better result, supporting our hypothesis that the motA mutation disrupts expression of motB.

The distance migrated when motA and motB were introduced into DS941 ΔmotA correlated well with the strength of the promoters driving expression of motA and motB. The two stronger promoters BBa_J23116 and BBa_J23106 restored swimming to a greater extent than the two weaker promoters BBa_J23103 and BBa_J23112 (Figure 2).

The motA motB J23100 promoter construct didn't give any colonies but ligations with other promoters did, suggesting that the J23100 promoter is too strong, and over expression of motility proteins could be toxic.


384px-GU_Gintare_Plates_3_small.png

Fig. 1: Swarm assay. 5µ drop of overnight culture was added on a soft-agar plate and left incubated overnight at 37°C. Both motA and motB under different strength promoters.

GU_Gintare_illustration_6.png

Fig. 2: DS941 ΔmotA E. coli carrying plasmids with the indicated biobricks were tested for mobility on swarm plates. Growth diameter of swarm assay grown for 16 hours at 37 degrees C. Non knock-out strain used as a control.



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
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 310

Functional Parameters

Protein data table for BioBrick BBa_K1463750 automatically created by the BioBrick-AutoAnnotator version 1.0
Nucleotide sequence in RFC 10: (underlined part encodes the protein)
 ATGAAGAAT ... GAACCGAGGTAA
 ORF from nucleotide position 1 to 924 (excluding stop-codon)
Amino acid sequence: (RFC 25 scars in shown in bold, other sequence features underlined; both given below)

101 
201 
301 
MKNQAHPIIVVKRRKAKSHGAAHGSWKIAYADFMTAMMAFFLVMWLISISSPKELIQIAEYFRTPLATAVTGGDRISNSESPIPGGGDDYTQSQGEVNKQ
PNIEELKKRMEQSRLRKLRGDLDQLIESDPKLRALRPHLKIDLVQEGLRIQIIDSQNRPMFRTGSADVEPYMRDILRAIAPVLNGIPNRISLSGHTDDFP
YASGEKGYSNWELSADRANASRRELMVGGLDSGKVLRVVGMAATMRLSDRGPDDAVNRRISLLVLNKQAEQAILHENAESQNEPVSALEKPEVAPQVSVP
TMPSAEPR*
Sequence features: (with their position in the amino acid sequence, see the list of supported features)
RFC25 scar (shown in bold): 163 to 164
Amino acid composition:
Ala (A)28 (9.1%)
Arg (R)25 (8.1%)
Asn (N)13 (4.2%)
Asp (D)18 (5.8%)
Cys (C)0 (0.0%)
Gln (Q)14 (4.5%)
Glu (E)20 (6.5%)
Gly (G)20 (6.5%)
His (H)6 (1.9%)
Ile (I)21 (6.8%)
Leu (L)27 (8.8%)
Lys (K)16 (5.2%)
Met (M)12 (3.9%)
Phe (F)6 (1.9%)
Pro (P)20 (6.5%)
Ser (S)26 (8.4%)
Thr (T)9 (2.9%)
Trp (W)3 (1.0%)
Tyr (Y)6 (1.9%)
Val (V)18 (5.8%)
Amino acid counting
Total number:308
Positively charged (Arg+Lys):41 (13.3%)
Negatively charged (Asp+Glu):38 (12.3%)
Aromatic (Phe+His+Try+Tyr):21 (6.8%)
Biochemical parameters
Atomic composition:C1493H2425N441O453S12
Molecular mass [Da]:34186.1
Theoretical pI:9.00
Extinction coefficient at 280 nm [M-1 cm-1]:25440 / 25440 (all Cys red/ox)
Plot for hydrophobicity, charge, predicted secondary structure, solvent accessability, transmembrane helices and disulfid bridges 
Codon usage
Organism:E. coliB. subtilisS. cerevisiaeA. thalianaP. patensMammals
Codon quality (CAI):good (0.78)good (0.77)acceptable (0.57)good (0.63)good (0.79)good (0.71)
Alignments (obtained from PredictProtein.org)
SwissProt: -
TrEML:L1C665 (100% identity on 308 AAs), W3UZH1 (100% identity on 308 AAs), W2A383 (100% identity on 308 AAs), W1T8J6 (100% identity on 308 AAs), W1SYM8 (100% identity on 308 AAs), W1BM97 (100% identity on 308 AAs), W0ZS26 (100% identity on 308 AAs), V8F1C0 (100% identity on 308 AAs), V6PJF3 (100% identity on 308 AAs), V6NTC2 (100% identity on 308 AAs)
PDB: -
Predictions (obtained from PredictProtein.org)
Subcellular Localization (reliability in brackets)
Archaea:cytosol (89%)
Bacteria:secreted (36%)
Eukarya:secreted (16%)
Gene Ontology (reliability in brackets)
Molecular Function Ontology: -
Biological Process Ontology:GO:0006810 (9%), GO:0001539 (43%)
 
Predicted features:
Disulfid bridges: -
Transmembrane helices:31 to 48 going outwards
The BioBrick-AutoAnnotator was created by TU-Munich 2013 iGEM team. For more information please see the documentation.
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References

1. Stuart W. Reid, Mark C. Leake, Jennifer H. Chandler, Chien-Jung Lo, Judith P. Armitage, and Richard M. Berry. (2006). The maximum number of torque-generating units in the flagellar motor of Escherichia coli is at least 11. PNAS, (103). 8066-8071.

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Categories
Parameters
None