- Courtney Calhoun
- Alexandra Popinga
G.tigrina regenerative Hox gene DthoxC insertion into prokaryote E.coli / (by UNIamCloning)
DthoxC is one of several Hox genes, which make up the homeobox domain, which
encodes for a homeodomain protein. The DthoxC gene regulates expression along
the anteroposterior axis during bilateral regeneration in the planarian Dugesia (Griardia) trigrina. The Hox gene’s functionality is not testable in E.Coli, so an SDS-
PAGE will need to be performed at the end of experimentation to show whether the
transfer and cloning of DthoxC was a success.
The DNA nucleotide sequence as well as the sequence for the amino acid translation
product were located using the NCBI gene bank: accession X95416.1. The DNA
sequence consists of a total of 942 base pairs and contains no introns.
To clone the DthoxC gene, DNA will be extracted from Dugesia trigrina and a
purification will be performed to clean up the sample. Then the gene will be
amplified via PCR using flanking primers specially designed for the DthoxC
sequence. The initial set of primers will be constructed using BioBrick-compatible
ends. If these fail, a second set will be created without the BioBrick extensions.
Once successfully amplified, the gene fragments and vectors will be digested using
BioBrick enzymes and the fragments will be spliced directly into the vector. There are also three EcoRI sites within the DthoxC gene which must be trimmed to avoid excess cutting by the restriction enzymes. The first two sites are space 18 base pairs apart, thus allowing an oligo of same or slightly greater length to account for both simultaneously. A couple of base pairs from the original nucleic sequence will be altered so that the enzymes will no longer be able to recognize and cut at the EcoRI sites.
Protein expression vectors will be used for this cloning process because Hox
gene functionality cannot be tested for in bacteria and protein expression will be
required for testing the effectiveness of the cloning process.
The plasmid vectors, with newly spliced DthoxC gene fragments, will be
transformed into chemically competent E.Coli bacteria. The bacteria will be grown
on plates, after which the plasmid DNA will be isolated and sequenced to test for the
presence of DthoxC gene fragments.
Once determined that the DthoxC gene has been inserted successfully, it will be
allowed to undergo transcription and translation, and with the addition of a strong
promoter, its expression - (homeodomain protein EMBL accession number: CAA64696.1) - will be tested via SDS-PAGE, or sodium dodecyl sulfate
polyacrylamide gel electrophoresis.
Homeodomain protein EMBL accession number: CAA64696.1. /
Promoters: Part:BBa_K215000 (R0011+B0034, strong IPTG-inducible promoter with strong RBS), Part:BBa_K320002, Part:BBa_K137029
Primers: 5'-ATG CAT GTC GAT CCT AAC GT-3' / 5'-CTA ATT GTA CTG CTG CGA GA-3'
BioBrick extensions: 5'-GAA TTC GCG GCC GCT TCT AG-3' prefix / 5'-GAA TTC GCG GCC GCT TCT AGA G-3' suffix
DNA extraction details: planarians starved for 1 week (Group A) and 2 weeks (Group B), homogenization in lysis buffer containing 100mM Tris*HCl, pH 7.4/100 mM EDTA/0.1% SDS, incubated with 100 ug/ml proteinase K for 5 hr at 50°C. Extract with phenol, phenol/chloroform, and chloroform, ethanol-precipitated in the presence of 0.3 M NaAc. Treat after resuspension with RNaseA (100 ug/ml)?
(Balavoine & Telford, 1995.  )
Powerpoint presentation: 
Primary research article: [http://dev.biologists.org/content/124/1/
Important Results and Milestones