Difference between revisions of "Part:BBa K2148001"
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===Verification=== | ===Verification=== | ||
Revision as of 11:58, 14 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 and is functional 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 was shot into CW15 strain of Chlamydomonas reinhardti and conferred resistance to kanamycin.
The part was also verified in E. coli, 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 652
- 1000COMPATIBLE 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/