Difference between revisions of "Part:BBa K1463704"

 
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K1463704 short</partinfo>
 
<partinfo>BBa_K1463704 short</partinfo>
  
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MotA and B0032 RBS under J23106 promoter.
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MotA with B0032 RBS under J23106 promoter.
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<!-- Add more about the biology of this part here
 
 
===Usage and Biology===
 
===Usage and Biology===
  
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We inserted the BBa_B0032 RBS – motA biobrick into the BBa_J61002 vector containing a variety of different promoters from the parts distribution:<BR>
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BBa_J23106 (½ the strength of J23100)<BR>
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BBa_J23116 (¼ the strength of J23100)<BR>
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BBa_J23103 (very weak promoter)<BR>
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BBa_J23112 (weakest promoter we could find in the registry, barely any expression)<BR>
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(Strength measured with RFP: Part BBa_J23100)
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These were checked by DNA sequencing and all found to have the expected sequence.
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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 Figure 1. DS941 ΔmotA did not swim at all, as expected for this mutant knocked out for the MotA motor protein. None of the plasmids containing just motA restored swimming to the mutant to any significant extent. <BR>
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Interestingly, however, composite biobricks containing motA and motB did restore swimming to our motA deletion mutant.
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See [https://parts.igem.org/Part:BBa_K1463700 BBa_K1463700] for more details.
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https://static.igem.org/mediawiki/2014/thumb/a/a9/GU_Gintare_Plates_2_small.png/384px-GU_Gintare_Plates_2_small.png
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'''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.
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https://static.igem.org/mediawiki/2014/2/27/GU_Gintare_illustration_5.png
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'''Fig. 5:''' DS941 ΔmotA E. coli carrying plasmids with the indicated biobricks were tested
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for mobility on swarm plates. Growth diameter of swarm assay grown for
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16 hours at 37 degrees C. Non knock-out strain used as a control.
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More information on the biobrick and usage:
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http://2014.igem.org/wiki/index.php?title=Team:Glasgow/Project/Mobility_Proteins#motA
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https://parts.igem.org/Part:BBa_K1463700
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K1463704 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1463704 SequenceAndFeatures</partinfo>

Latest revision as of 20:46, 23 October 2014

MotA and B0032 RBS under J23106 promoter

MotA with B0032 RBS under J23106 promoter.


Usage and Biology

We inserted the BBa_B0032 RBS – motA 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)

These 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 Figure 1. DS941 ΔmotA did not swim at all, as expected for this mutant knocked out for the MotA motor protein. None of the plasmids containing just motA restored swimming to the mutant to any significant extent.

Interestingly, however, composite biobricks containing motA and motB did restore swimming to our motA deletion mutant. See BBa_K1463700 for more details.


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.



More information on the biobrick and usage:

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

https://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