Coding
motA

Part:BBa_K1463700

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

MotA

MotA 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_K777113. Changes made can be found in the design section.


F1.medium.gif

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


Usage and Biology

Our MotA biobrick with B0032 RBS (making the composite biobrick BBa_K1463701) was ligated into the pSB1C3 submission vector and also the plasmid J61002 containing the strong J23100 promoter. After transformation colonies were only obtained with the pSB1C3 vector. A repeated ligation into the vector containing the strong J23100 promoter gave two colonies. However, sequencing showed that while motA clones in pSB1C3 had the correct sequence, the two inserts downstream of BBa_J23100 had mutations. One contained a mutation in the ribosome binding site, while the other had a 5 base deletion at the 5' end of the motA gene. This suggested that promoter J23100 was too strong and that high levels of motA expression might be toxic to the cells.

We inserted the B0032 RBS – motA composite biobrick BBa_K1463701 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)

These constructs (corresponding to composite biobricks BBa_K1463703, BBa_K1463704, BBa_K1463705, and BBa_K1463706) were checked by DNA sequencing and all found to have the expected sequence.

We then used swarm assays (semi-solid agar motility test) to investigate whether these plasmids would rescue swimming of a motA mutant. DS941 ΔmotA (with motA deleted) was transformed with pSB1C3 motA (no promoter), motA transcribed from the four different weaker promoters in BBa_J61002, and also motA with the strong J23100 promoter with the mutated ribosome binding site. The results of the swarm assays are shown in Figures 1 and 3. DS941 and MG1655-Z1 (another positive swimming control) swam to approximately the same distance. Three different isolates of DS941 ΔmotA did not swim at all, as expected for this mutant knocked out for the MotA motor protein. However, none of the plasmids containing motA restored swimming to the mutant to any significant extent, although it is possible that pSB1C3-motA (with no promoter) and BBa_J23100 – motA plasmid (with mutant RBS) gave slightly more mobility than no plasmid at all (Figures 1 and 3).

MotA is expressed from an operon containing two flagellar motor genes, motA and motB, and both of these genes are required for motor function, and hence swimming. Deletions in upstream genes in operons are often known to have “polar” effects, disrupting expression of downstream genes. Therefore our motA deletion might be severely reducing expression of motB. To test this, we made a motA-motB biobrick and check whether it restores swimming to our ΔmotA mutant.

The motA motB J23100 promoter construct didn't give any colonies but ligations with other promoters did, suggesting again that the J23100 promoter is too strong, and over expression of motility proteins could be toxic. DS941 ΔmotA was transformed with BBa_J23103 motA motB, BBa_J23106 motA motB, BBa_J23112 motA motB and BBa_J23116 motA motB (corresponding to composite biobricks BBa_K1463770, BBa_K1463771, BBa_K1463772, and BBa_K1463773) all in the plasmid vector BBa_J61002. Gene rescue was checked again by doing swarm assay (Figure 5). This time we saw a significantly better result than just with motA, supporting our hypothesis that the motA mutation disrupts expression of motB.

The diameter of migration on the swarm plates is shown in the histograms in figure 2, 4 and 6. 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.


288px-GU_Gintare_Plates_1_Small.png

Fig. 2: Swarm assay. 5µ drop of overnight culture was added on a soft-agar plate and left incubated overnight at 37°C.

GU_Gintare_illutration_4.png

Fig. 3: 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 strains used as a controls.

384px-GU_Gintare_Plates_2_small.png

Fig. 4: Swarm assay. 5µ drop of overnight culture was added on a soft-agar plate and left incubated overnight at 37°C. Different strength promoters used this time.

GU_Gintare_illustration_5.png

Fig. 5: 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.

384px-GU_Gintare_Plates_3_small.png

Fig. 6: 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. 7: 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. The first column shows DS941, a non knock-out strain as control. The other columns show the motA knockout mutant (DS941 ΔmotA) with plasmids carrying motA and motB driven by different strength constitutive promoters.



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 323
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 61
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 829

Functional Parameters

Protein data table for BioBrick BBa_K1463700 automatically created by the BioBrick-AutoAnnotator version 1.0
Nucleotide sequence in RFC 10: (underlined part encodes the protein)
 ATGCTTATC ... GAGGAAGCATAA
 ORF from nucleotide position 1 to 885 (excluding stop-codon)
Amino acid sequence: (RFC 25 scars in shown in bold, other sequence features underlined; both given below)

101 
201 
MLILLGYLVVLGTVFGGYLMTGGSLGALYQPAELVIIAGAGIGSFIVGNNGKAIKGTLKALPLLFRRSKYTKAMYMDLLALLYRLMAKSRQMGMFSLERD
IENPRESEIFASYPRILADSVMLDFIVDYLRLIISGHMNTFEIEALMDEEIETHESEAEVPANSLALVGDSLPAFGIVAAVMGVVHALGSADRPAAELGA
LIAHAMVGTFLGILLAYGFISPLATVLRQKSAETSKMMQCVKVTLLSNLNGYAPPIAVEFGRKTLYSSERPSFIELEEHVRAVKNPQQQTTTEEA*
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)32 (10.8%)
Arg (R)13 (4.4%)
Asn (N)8 (2.7%)
Asp (D)8 (2.7%)
Cys (C)1 (0.3%)
Gln (Q)7 (2.4%)
Glu (E)22 (7.5%)
Gly (G)25 (8.5%)
His (H)5 (1.7%)
Ile (I)20 (6.8%)
Leu (L)40 (13.6%)
Lys (K)11 (3.7%)
Met (M)14 (4.7%)
Phe (F)12 (4.1%)
Pro (P)12 (4.1%)
Ser (S)20 (6.8%)
Thr (T)14 (4.7%)
Trp (W)0 (0.0%)
Tyr (Y)11 (3.7%)
Val (V)20 (6.8%)
Amino acid counting
Total number:295
Positively charged (Arg+Lys):24 (8.1%)
Negatively charged (Asp+Glu):30 (10.2%)
Aromatic (Phe+His+Try+Tyr):28 (9.5%)
Biochemical parameters
Atomic composition:C1445H2318N370O416S15
Molecular mass [Da]:32011.4
Theoretical pI:5.47
Extinction coefficient at 280 nm [M-1 cm-1]:16390 / 16453 (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 (0.79)acceptable (0.58)good (0.66)good (0.78)good (0.64)
Alignments (obtained from PredictProtein.org)
SwissProt:P55891 (93% identity on 295 AAs)
TrEML:W7HZV0 (99% identity on 295 AAs), W1T4P9 (99% identity on 295 AAs), W0ZTN4 (99% identity on 295 AAs), W0KXY2 (99% identity on 295 AAs), W0KCE0 (99% identity on 295 AAs), V8KS87 (99% identity on 295 AAs), V8FL71 (99% identity on 295 AAs), V8F085 (99% identity on 295 AAs), V6PL48 (99% identity on 295 AAs), V6NU46 (99% identity on 295 AAs)
PDB: -
Predictions (obtained from PredictProtein.org)
Subcellular Localization (reliability in brackets)
Archaea:plasmamembrane (61%)
Bacteria:innermembrane (100%)
Eukarya:golgimembrane (22%)
Gene Ontology (reliability in brackets)
Molecular Function Ontology:GO:0042802 (46%)
Biological Process Ontology:GO:0015992 (35%), GO:0001539 (49%)
 
Predicted features:
Disulfid bridges: -
Transmembrane helices:3 to 20 going outwards, 32 to 49 going inwards, 171 to 188 going outwards, 202 to 219 going inwards
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.

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.

[edit]
Categories
Parameters
None