Monica's Barrett Presentation

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  • Over the summer I was able to participate in a research project based on a bacterium that can produce cyanobacteriochromes. The Thermosynechococcuselongatus. Particularly, how protein expression works with a certain gene, which I will explain.
  • If you remember from the previous presentation, Stefanie, Kristen and Alex explained how we capture and truncation the DNA sequence, construct a plasmid with our target sequence, site directed mutagenesis. Today Mo, and I are going to explain how we express our engineered DNA sequence as protein. Then Valerie will talk about how to purified the protein away from out E coli protein making system.T elongatus is a cyanobacterium: major group of photosynthetic bacteria; they are prokaryotic (O2-evolving type of photosynthesis resembling that of green plants and contain chlorophyll a and phycobilin pigments.These bacterium make cyanobacteriochromes (one protein of many that they make) – they are light sensitive proteinsThey are thermophilic proteins (protein structures are stable in high temperatures; strong bonds to keep shape; use this type of protein a lot to experiment with) which are photoreversible (absorbs two different wavelengths of light to assume an active and inactive form directing bacterium to respond appropriately in various light environments).Phytochromes are light sensitive proteins in plants (detectors); Pr are red light sensitive which converts to Pfr which are far red light sensitive. There are two photosystems (I and II) that are homologous and found in all five genes in T. elongatus. PSI and PSII can be crystallized for x-ray diffraction (much like DNA was crystallized by Rosalind Franklin to determine its double helix strands).
  • Overview of project - HIV-infected Jurkat Lymphocyte Cells are filmed using a spinning disk light microscope at The Center for Biophotonics at U.C. Davis. The microscope is adapted for viewing fluorescence. We are planning to use our red tags to illuminate the actin or tubulin cytoskeleton. This may reveal pathways for traffic control during virus infection.
  • Working on these genes for ten years with support from CBST; continuing work of many researchers who have contributed over the past ten years.I was working with plasmids that were previously constructed.We truncate gene of huge sequence to gafdomian (pcr to truncate, then use restriction enzymes to insert into vector plasmid called pBAD).
  • We have the stock of E.coli (LMG194) that contains pPL plasmids. The pPL plasmids contain two enzymes that will make our bilinchromophore in the E.coli, from the E.coli’s own supply of HEME, After transformation, e.coli has two plasmids, pBAD and pPL. They are under the control of chemicals IPTG and L-arbonose. LMG194 will express from both plasmids when these chemicals are added. As the protein that we engineered is expressed, it will covalently bind the bilin, which is made from the E.coli’s HEME. When the protein binds to the bilin, it becomes sensitive/reactive to the photons. Whenpbad and ppl are added together, they produce the gaf domain and pcb binding pocket which are both needed for expression.
  • Discovered something useful about 911 this summer: 911T was really 569TM. We kept getting blue pellets, but we knew they should’ve been yellow. So then we sequenced the pellets after purifying them (as Valerie will describe in detail) and the results concluded that the pellets were the mutated form of another gene in T elongatus. We will sequence the yellow pellets and future pellets and hopefully they will be 911T.
  • Monica's Barrett Presentation

    1. 1. Bioengineering fluorescent protein tags from cyanobacteriochromes from T.elongatus: Protein Expression<br />CBST RESEARCH PROJECT<br />SUMMER 2011<br />PRESENTED BY Monica bower<br />WITH SUPPORT FROM <br />DR. SUSAN C. SPILLER, <br />And members of the spiller lab<br />
    2. 2. T. elongatus BP-1<br /><ul><li>Origin: Japan in Beppu Hot Springs (BP-1)
    3. 3. Cyanobacterium
    4. 4. Group of photosynthetic bacteria
    5. 5. Prokaryotic
    6. 6. Chlorophyll a + phycobilin pigments
    7. 7. T. elongatusin particular
    8. 8. Genes make cyanobacteriochromes
    9. 9. Thermophilic
    10. 10. Photoreversibility
    11. 11. Cyanobacteriochromes – blue/green
    12. 12. Engineered cyanobacteriochromes – red fluorescent</li></li></ul><li>Imagine the recipient cells showing their actin or tubulincyto-skeletons in bright red!<br />
    13. 13. T. elongatus Genes<br />MINE!<br />
    14. 14. Two Plasmid System<br />pPL plasmid<br />Contains genetic information to make PCB<br />Hemoxygenase (Ho1)<br />Reductase (PcyA)<br />
    15. 15. LMG194 – PCB E. coli <br />(outer layer)<br />pPL plasmid<br />pBAD plasmid<br />KanR<br />Proteins<br />AmpR<br />Plates with colonies and both Ampicillin and Kanamycin<br />
    16. 16. 1 2 3 4<br />Photos: Tami Port, MS<br />Proteins<br />2 mL culture = RM broth (has glucose to turn off lac-operon) + billions of bacteria w/ plasmids <br />500 mL = LB broth + IPTG (turns on pPL plasmid) – after 1 hour: add L-ara (turns on pBAD plasmid)<br />50 mL = RM broth + ampicillin + kanamycin + bacteria<br />
    17. 17. Protein Expression<br />Blue?!<br />Can’t be 911T.<br />Yellow! Could be 911T…<br />Proteins<br />911: Photoreverses to appear yellow<br />Proteins<br />

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