Difference between revisions of "Part:BBa K2148001"

(→‎Verification)
 
(2 intermediate revisions by 2 users not shown)
Line 7: Line 7:
  
 
===Usage and Biology===
 
===Usage and Biology===
−
The aadA gene confers antibiotic resistance to Spectinomycin and Streptomycin and is functional in both chloroplasts and <i>E. coli</i>.  
+
The aadA gene confers antibiotic resistance to Spectinomycin and Streptomycin in both chloroplasts and <i>E. coli</i>.  
  
 
This part can be used with other level 0 Phytobrick to create a transcriptional unit to provide a selection pressure on transformations.  
 
This part can be used with other level 0 Phytobrick to create a transcriptional unit to provide a selection pressure on transformations.  
Line 15: Line 15:
 
Has been successfully used to construct LV1 constructs through the golden gate protocol thus establishing the phytobrick standard functionality.  
 
Has been successfully used to construct LV1 constructs through the golden gate protocol thus establishing the phytobrick standard functionality.  
  
−
===Verification===
+
<html>
 +
    <figure>
 +
        <img src="https://static.igem.org/mediawiki/2016/2/22/T--Cambridge-JIC--description1.png" width="" height="450" style="float:center">
 +
        <figcaption>
 +
        <p style="font-size:11px">
 +
        </p>
 +
        </figcaption>
 +
    </figure>
 +
</html>
  
−
aadA was assembled with HomR-psaA promoter/5UTR/NTAG and rbcL 3UTR/TERM-HomL to create a functional gene unit that was shot into CW15 strain of <i>Chlamydomonas reinhardti</i> and conferred resistance to kanamycin.
 
  
−
The part was also verified in <i>E. coli</i>, ligated into Paul Surton's (UCL) pASap1 backbone. These transformed bacteria grow on both ampicilin (backbone antibioti) and spectinomycin (albeit slowly). Picture below indicates growth on Spec plates (before plating on amplicilin plates to verify double resistance).  
+
===Verification===
 +
 
 +
aadA was assembled with HomR-psaA promoter/5UTR/NTAG and rbcL 3UTR/TERM-HomL to create a functional gene unit that conferred resistance to spectinomycin in <i>E. coli</i>, whose protein expression system is similar to <i>Chlamydomonas reinhardtii</i> chloroplast. It ligated into Paul Surton's (UCL) pASap1 backbone. These transformed bacteria grow on both ampicilin (backbone antibioti) and spectinomycin (albeit slowly). Picture below indicates growth on Spec plates (before plating on amplicilin plates to verify double resistance).  
  
 
<html>
 
<html>

Latest revision as of 21:07, 19 October 2016


aadA

A commonly used antibiotic for Chlamydomonas chloroplasts. Confers resistance to spectinomycin and streptomycin.


Usage and Biology

The aadA gene confers antibiotic resistance to Spectinomycin and Streptomycin in both chloroplasts and E. coli.

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

Previous experience with this antibiotic marker in Saul Purton's lab recommends the psaA promoter, which is a strong promoter. However, when constructing level 2 composite parts, it is advisable to use the psaA promoter for the gene of interest and place aadA under atpA promoter which is weaker.

Has been successfully used to construct LV1 constructs through the golden gate protocol thus establishing the phytobrick standard functionality.


Verification

aadA was assembled with HomR-psaA promoter/5UTR/NTAG and rbcL 3UTR/TERM-HomL to create a functional gene unit that conferred resistance to spectinomycin in E. coli, whose protein expression system is similar to Chlamydomonas reinhardtii chloroplast. It ligated into Paul Surton's (UCL) pASap1 backbone. These transformed bacteria grow on both ampicilin (backbone antibioti) and spectinomycin (albeit slowly). Picture below indicates growth on Spec plates (before plating on amplicilin plates to verify double resistance).

Sequence

Sequencing data obtained from Biosource sequencing, the quality is worse at the 3' end (note N symbolizes un-indentified nucleotide) ATTTNACGTAGTGGACAAATTCTTCCNACTGATCTGCGCGCGAGGCCAAGCGATCTTCTTCTTGTCCAAGATAAGCCTGTCTAGC TTCAAGTATGACGGGCTGATACTGGGCCGGCAGGCGCTCCATTGCCCAGTCGGCAGCGACATCCTTCGGCGCGATTTTGCCGGTT ACTGCGCTGTACCAAATGCGGGACAACGTAAGCACTACATTTCGCTCATCGCCAGCCCAGTCGGGCGGCGAGTTCCATAGCGTTA AGGTTTCATTTAGCGCCTCAAATAGATCCTGTTCAGGAACCGGATCAAAGAGTTCCTCCGCCGCTGGACCTACCAAGGCAACGCT ATGTTCTCTTGCTTTTGTCAGCAAGATAGCCAGATCAATGTCGATCGTGGCTGGCTCGAAGATACCTGCAAGAATGTCATTGCGC TGCCATTCTCCAAATTGCAGTTCGCGCTTANCTGGATAACGCCACGGAATGATGTCGTCGTGCACAACAATGGTGACTTCTACAG CGCGGAGAATCTCGCTCTCTCCAGGGGAAGCCGAAGTTTCCAAAANGTCGTTGATCAAAGCTCGCCGCGTTGTTTCATCAAGCCT TANGTCACCGTAACCAGCAGATCAATATCACTGTGTGGCTTCNNCCGCCATCCACTGCGGAGCCGTACAAATGTACGGCCAGCAA CGTCNTTCGAGATGGCGCTCGATGACGCCAACTACCTCTGATANTTGAGTTGANACTTCGGCGATACCGCTTCACGANCCATTCG AGACCACTCNTCGCTNCTAAANANNGGCCNCAATTCCNNNNNTCNNNNNNNCCNAANGCTAAGGATTTTNTTAATCTGAAATTCN NGCCTNNNGATACNCTAATTTTANAGGTAATGNCANNNNNANNNNNCNNNNANN


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 652
  • 1000
    COMPATIBLE WITH RFC[1000]


References

Goldschimdt-Clermont, M.: Transgenic expression of aminoglycoside adenine transferase in the chloroplast: a selectable marker of site-directed transformation of chlamydomonas. Nucleic Acids Res. 1991 Aug 11; 19(15): 4083–4089. Online at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC328544/