Difference between revisions of "Part:BBa K2148002"
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Selection conditions are more difficult for this antibiotic resistance gene. The construct should be functional in <i>E. coli</i> conferring kanamycin resistance @20µg/ml, observations indicate that the marker doesn't usually work well in the initial round of selection for positive transformants. The recommended strategy is to first select for <i>E. coli</i> transformants using the marker carried in the plasmid backbone (chloramphenicol for level 0 parts, kanamycin for level 1 part "alphas"  and spectinomycin/streptinomycin for level 2 part "omegas"). Then the transformant colonies can be scored on 20µg/ml kanamycin plates.     
 
Selection conditions are more difficult for this antibiotic resistance gene. The construct should be functional in <i>E. coli</i> conferring kanamycin resistance @20µg/ml, observations indicate that the marker doesn't usually work well in the initial round of selection for positive transformants. The recommended strategy is to first select for <i>E. coli</i> transformants using the marker carried in the plasmid backbone (chloramphenicol for level 0 parts, kanamycin for level 1 part "alphas"  and spectinomycin/streptinomycin for level 2 part "omegas"). Then the transformant colonies can be scored on 20µg/ml kanamycin plates.     
  
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===Use in <i>Chlamydomonas reinhardtii</i> chloroplasts
+
===Use in <i>Chlamydomonas reinhardtii</i> chloroplasts===
 
Cambridge-JIC 2016 team have developed a strategy that should, in theory, accelerate the process of achieving homoplasmy -- one of the most time-consuming stages of plastid transformation. This would depend on the double-transformation with two different antibiotic resistance genes, one with can be aphA6, if expressed from the psaA promoter from our library. The second can be aadA, which we are also submitting.
 
Cambridge-JIC 2016 team have developed a strategy that should, in theory, accelerate the process of achieving homoplasmy -- one of the most time-consuming stages of plastid transformation. This would depend on the double-transformation with two different antibiotic resistance genes, one with can be aphA6, if expressed from the psaA promoter from our library. The second can be aadA, which we are also submitting.
  

Revision as of 18:04, 13 October 2016


aphA6

This part encodes the enzyme aminoglycoside phosphotransferase which confers antibiotic resistance to amikacin, neomycin, and kanamycin. The CDS is flanked by Phytobrick fusion sites which corresponds to a CDS/CTAG Phytobrick.

Usage and Biology

This part can be used with other level 0 Phytobrick to create a transcriptional unit to provide a selection pressure on transformations.

Selection conditions are more difficult for this antibiotic resistance gene. The construct should be functional in E. coli conferring kanamycin resistance @20µg/ml, observations indicate that the marker doesn't usually work well in the initial round of selection for positive transformants. The recommended strategy is to first select for E. coli transformants using the marker carried in the plasmid backbone (chloramphenicol for level 0 parts, kanamycin for level 1 part "alphas" and spectinomycin/streptinomycin for level 2 part "omegas"). Then the transformant colonies can be scored on 20µg/ml kanamycin plates.

Use in Chlamydomonas reinhardtii chloroplasts

Cambridge-JIC 2016 team have developed a strategy that should, in theory, accelerate the process of achieving homoplasmy -- one of the most time-consuming stages of plastid transformation. This would depend on the double-transformation with two different antibiotic resistance genes, one with can be aphA6, if expressed from the psaA promoter from our library. The second can be aadA, which we are also submitting.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 647
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 647
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 647
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 647
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 647
  • 1000
    COMPATIBLE WITH RFC[1000]


Verification

Part verified into the level 0 iGEM phytobrick backbone through restriction digest where the band produced was of the expected size, see gel image below. This part is labelled AP1 and the molecular ladder is Bioline 1kB.

INSERT GEL IMAGE HERE

References

Bateman J. M., Purton S. (2000). Tools for chloroplast transformation in Chlamydomonas: expression vectors and a new dominant selectable marker. Mol. Gen. Genet. 263, 404–410. 10.1007/s004380051184