S-STEM Scholar Leilani Boren's Blog

 Introduction: In the previous weeks, the fragments of left, tetr/a, and right have been isolated for overlap PCR and identical fragments for RE ligation were beginning to be processed. This week consisted of successfully isolating RE fragments, as well as cleaning all fragments for both methods. Then digestion was started for RE fragments while overlap binding was attempted for the left and tetr/a fragments with PCR.  Methods:  All amplifying PCR reactions with a 20 ul total volume consisted of 16 ul of Mastermix, 2 ul of DNA template, and 1 ul of each primer. Alll reactions with a volume of 50 ul were done with 43 ul of Mastermix, 2 ul of DNA template, and 2.5 ul of primers. Gels were consistently 1x TAE 1% agarose with a 5 ul of sample loaded for each lane.   The RE fragments were isolated in 20 ul PCR reactions with freshly made Mastermix to correct the lack of results from the previous week's gels. Each sample was processed in the standard Monarch PCR clean...

Introduction:     From the previous week, two of three fragments needed for linear plasmid construction in order to successfully transform Deinococcus radiodruans  had been visualized on an agarose gel for overlap PCR. This week, it was decided to also pursue a second route in plasmid construction, restriction enzyme ligation. By dividing into two groups the team will be able to separately test overlap PCR and RE ligation techniques and evaluate differences between the two methods in terms of time, resources, 0=d obstacles. The fragments will be independently isolated with RE ligation specific primers to move forward in that direction.  Methods:     Deinococcus radiodurans was isolated from Mueller-Hinton broth and DNA extraction was completed using the Ultraclean DNeasy extraction kit for template PCR DNA. E. coli  was also inoculated in LB broth and grown statically at 37 degrees Celsius to acquire more template plasmid DNA using a Monarch plasmid ex...

 Introduction:   Deinococcus radiodurans   or D. rad  is known for being an extremophilic bacterial species that can withstand high levels of oxidative stress and is incredibly efficient when repairing chromosomal DNA breakages. This bacterial species has been widely studied as of late due to these abilities, but it is the species of interest for this project for its potential uses in the future.  D. rad  chromosomal DNA contains genes for an AMC pathway to metabolize methionine. A part of the pathway, the LuxS gene, is critical for the pathway and is suspected to be integral for AHL production and maintenance.  D. rad  is also a species that no known linear plasmids currently exist in nature. The goal of this project is to knock out the LuxS gene of the AMC pathway by transforming  D. rad  with a linear plasmid consisting of three fragments (left, right, and tetr/a) which will replace the LuxS gene in the circular DNA and produce a bact...