Composite

Part:BBa_K1463772

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

MotA and MotB under J23106 Promoter

MotA and MotB, both with B0032 RBS, under J23106 promoter.


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.


For more information on the biobrick and usage go to:

http://2014.igem.org/wiki/index.php?title=Team:Glasgow/Project/Mobility_Proteins#motA

http://parts.igem.org/Part:BBa_K1463750

http://parts.igem.org/Part:BBa_K1463700



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 385
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 123
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 891
    Illegal SapI.rc site found at 1287
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