Difference between revisions of "Part:BBa K2997009"

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Fig. 6. Confirmation of BBa_K2997009 by double digestion, arrow indicates TAL w/ NRBS (~1600bp).M: Marker; Lane 1: pSB1C3-BBa_K2997009; Lane 2: BBa_K2997009
 
Fig. 6. Confirmation of BBa_K2997009 by double digestion, arrow indicates TAL w/ NRBS (~1600bp).M: Marker; Lane 1: pSB1C3-BBa_K2997009; Lane 2: BBa_K2997009
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===RT-PCR===
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RT-PCR experiment was used to confirm that the constructed TAL Biobrick is being transcribed. As seen in Fig.9, cDNA for both bacteria carrying TAL constructs are being detected by PCR. Confirming that the TAL genes is actually being transcribed in E. coli Nissle.
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 17:44, 19 October 2019


J23100-NRBS-sam8 (J23100-I742106)

Background

Tyrosine ammonia-lyase (TAL) is an enzyme that converts tyrosine into p-Coumaric acid. We engineered E. coli Nissle to express TAL to turn the excess tyrosine inside the gut into p-Coumaric acid, and compare the different constructs for improvement, BBa_K2997009 and BBa_K2997010.

Expression in E.coli

We constructed Tyrosine Ammonia Lyase with a native RBS , and transformed the plasmid into E. coli Nissle 1917 and confirmed it by double digestion. The results are as follows:


Fig. 6. Confirmation of BBa_K2997009 by double digestion, arrow indicates TAL w/ NRBS (~1600bp).M: Marker; Lane 1: pSB1C3-BBa_K2997009; Lane 2: BBa_K2997009

RT-PCR

RT-PCR experiment was used to confirm that the constructed TAL Biobrick is being transcribed. As seen in Fig.9, cDNA for both bacteria carrying TAL constructs are being detected by PCR. Confirming that the TAL genes is actually being transcribed in E. coli Nissle.




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 457
    Illegal BamHI site found at 36
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 232