lab notebook

Overview

CAP’siRNA aimed to engineer bacteria to produce precursors of RNA interference, long hairpin RNA (lhRNA). RNA being unstable in the environment, we planned to encapsulate them in the capsid of the Tobacco Mosaic Virus (TMV) to protect them and allow their entry into the plants. To obtain the coat proteins of the TMV, we wanted to try two approachs: isolate them from the TMV directly or produce them in bacteria and isolate them from there.
Therefore, we organized our work in the laboratory in three parts:

• lhRNA engineering
• Coat proteins isolation
• Coat proteins engineering

Week 1

• The TMV was mechanically inoculated to our sugar beets

• The backbone pET21a(+) was amplified by transformation in DH5a followed by miniculture and MiniPrep

Week 2

• The coat proteins were isolated from the sugar beets and purified

• The backbone pET21a(+) and the insert sequences were digested with the corresponding restriction enzymes

Week 3

• The pET21a(+) was ligated with the PDS inserts

• The pET21a(+) was reamplified by transformation in DH5a and MiniPrep

• The pET21a(+) was digested again with restriction enzymes

Week 4

• The isolated coat proteins were quantified using BCA assay

• TMV was inoculated again in sugar beets

• A plasmid encoding mCherry was transformed into E. coli DH5a

• The golden gate backbone (AF_lacZ pDest) was transformed into E. coli DH5a

Week 5

• pET21a(+) was again Minipreped and digested with restriction enzymes

• The PDS inserts were ligated with the pET21a(+) again and the transformation product was transformed in E. coli DH5a

• The different plasmids to use for the golden gate were transformed into E. coli DH5a and isolated using MiniPrep

Week 6

• The coat proteins were purified again from frozen infected leaves

• The isolated proteins were quantified using BCA assay again

• The isolated proteins were separated with SDS-PAGE

• PCR was performed on different inserts

• The pET21a(+) was ligated with the p21 inserts and the ligation product was transformed in E. coli DH5a

• pET21a(+) was amplified again

• Different control for the ligations were made

• Different RBSs were transformed into E. coli DH5a and MiniPreped

Week 7

• The coat proteins were precipitated and quantify by BCA assay

• TMV was inoculated again in sugar beets

• The pET21a(+) and the inserts were digested again

• Partial and total ligations of the different inserts with the backbone were attempted and transformed in E. coli DH5a

• The golden gate sequences of interest were amplified with PCRs

Week 8

• The inserts were re-digested and ligated together and with the pET21a(+) with different combinations

• The inserts were also digested with alkaline phosphatase

• The pET21a(+) was MiniPreped

Week 9

• TMV coat protein were isolated

• New potential backbones were digested: pET22 and pET11

• The inserts were independently inserted in a plasmid to be amplified

• RNA was extracted from saine and infected leaves

Week 10

• TMV coat protein were quantified

• The different inserts were ligated in plasmid to amplify them

• RT-qPCR was performed on the RNA extracted from saine and infected sugar beet leaves

Week 11

• SDS-PAGEs were performed to confirm coat proteins isolation

• The different inserts were digested and ligated with the new backbones

• RT-qPCR was performed on the RNA extracted from saine and infected sugar beet leaves

• The basic parts to assemble with Golden Gate were amplified by PCR

• Golden Gate assembly was performed