03.08.2015   BIOFUEL'S ENGINEERING PROCESS TECHNOLOGY

Molecular cloning and construction of vectors

All plasmids were prepared and isolated from bacterial hosts using the QIAPrep Kit (Qiagen) following the supplier’s protocol. E. coli DH5a cells (Invitrogen) were grown axenically either on LB agar or in LB medium containing pertinent antibiotic selective pressure (ampicillin or kanamycin) at 37° C. Genomic DNA was extracted from D. salina using standard CTAB protocol adapted from Volvox carteri. PCR was performed using the MJ Mini (Bio-Rad) to amplify the actin (GenBank: AF541875) and rubisco small subunit (GenBank: AY960592) promoters and nitA 3′-UTR (GenBank: EF156403) from D. salina UTEX 1644 genomic DNA. Likewise, the genes ble and bar were amplified from the plasmids pSP124 and pGR117, respectively. All primers employed in this study are listed below in Table 2. Each PCR product was subsequently ligated into the subcloning plasmid pGEM®-T Easy (Promega) using T4 DNA Ligase and its corresponding buffer (Invitrogen). Sequencing of the genetic fragments derived from PCR was performed at the UMBC Biological Sciences Dept. DNA sequencing facility using BigDye® (Applied Biosystems).

Target Sequence

Primers (including NotI, SmaI, HindIII, and XhoI for subcloning)

actin promoter

5′-AATAATAGCGGCCGCCACGGCTCACCATCTTGTTT-3′

5′-AATAATACCCGGGTTGATCTCTCTGTCACCCCT-3′

rbcS2 promoter

5′-AATAATAAGCGGCCGCAGACATGAACCTATA-3′

5′-AATAATAACCCGGGAGGTCTTGGCAATGA-3′

bar

5′-AAT AAT ACCCGGG ATG AGCCCAGAACGACGCCC-3′ 5′-AATAATAAAGCTTTCAGATTTCGGTGACGGGCA-3′

ble

5′-AAT AAT ACCCGGG ATGTTCTTTACTTTTTTACA-3′ 5′-AAT AATAAAGCTTCTAGAGTGGGTCGACGTCGG-3′

aphVIII

5′-A AT AAT ACCCGGGCG AAGC ATGG ACGATGCGTT-3′ 5′-AATAATAAAGCTTTCAGAAGAACTCGTCCAACA-3′

nitA 3′-UTR

5′-AAT AATAAAGCTTGCGGGGTCAGCAGGAGCGAC-3′ 5′-AATAATACTCGAGTCGATCAGCCTTTGCAATCC-3′

Table 2. Primers used to amplify genes and promoters for vector development.