1. Researchers names and affiliations
Research Title
NSF HBCU-UP Program
Fort Valley State University
Researchers names and affiliations
Glass Bead Transformation
Results
Abstract
More than 7k are deleted from A10 genome causing 3 genes to be impacted. We extracted each gene from a bacterial artificial chromosome (BAC) vector and transformed them individually back into the mutant in order to restore the fatty acid phenotype. We then used gas chromatography in order to see if the gene complemented the mutant.
Genes one and two where transformed back into the A10 mutant using the glass bead transformation technique
The cells and DNA were vortexed for 15 seconds in a glass tube which contained 0.3 g of 0.4 mm glass beads.
The cells were then poured in tris acetate phosphate plates that contained the antibiotics hygromycin and ampicillin.
5 days after transformation.
Part of BAC plasmid with Restriction Sites
AvrII
MfeI
BamHI
Gene #1
Gene #2
Gene #3
1 day after transformation
Gene #1
Unknown Conserved Function
Only partially deleted
Gene #1 was extracted using a single Digestion with enzyme AvrII
Gene #2
Codes for an RNA-binding protien
Completely deleted
Gene # 2 was extracted using a double and single digestion with enzyme MfeI and enzymes MfeI paired with BamHI
Gene #3
Partially deleted
Only the 3’ UTR is affected.
For the #1 gene 42 putative transformants were tested and 10 were found to be positive co-transformants
C AT Wt
Deleted
Problems and Setbacks
Contamination
Introduction
#1 gene from AVRII single digestion
Triacylglycerol (TAG)
WT Chlamydomonas Reinhardtii
Non-polar lipid
Highly reduced form of carbon for storing energy
Capable of containing longer carbon chains than gasoline
Since we were unable to isolate the #3 gene we speculate that some random mutation may have occurred in the back vector which is why our enzymes didn’t work. The BAC vector is currently being sequenced.
DELETED
About A10 mutant
A10 has a low oil phenotype compared with mutants and wild type. And a lower growth rate than wild type.
MfeI and BamHI double digestion.
MfeI single digestion
Conclusion
Deleted
Four different double digestions were used to isolate the gene.
No digestion could successfully isolate the gene.
Acknowledgements
Future Research