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Stefanie yoshizuka pzazz

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  • pGLO is a plasmid that has been engineered to contain the Green Fluorescent Protein (GFP) gene, which was originally isolated from the jelly Aequoreavictoria. GFP produces a green fluorescence when excited by blue or UV light.
  • In this pGLO kit, the plasmids have been engineered to contain this GFP gene from jellyfish. So, what is a plasmid? Plasmids are circular double-stranded DNA of bacteria that work as “satellites” and are not part of their chromosome. This makes for a convenient tool for bioengineering because it’s a vector for moving selected sequences of DNA into a new cell. In this kit, you are given tools to transfer DNA from one organism to another (in this case, from jellyfish to E. coli). This DNA transfer makes for a great research and teaching tool. In thepGLO plasmid, in order to make the GFP gene functional, it has been engineered so that the sugar arabinose triggers the production of the protein. In this case, the genes in the arabinoseoperon have been replaced by the GFP gene where the regulatory sequence has been left intact. The presence of arabinose turns on the GFP gene, and therefore, GFP is produced. Here, is the origin of replication which allows for replication of the plasmid every time the bacteria divides. Additionally, the pGLO plasmid also contains the beta-lactamase gene which provides resistance against the antibiotic ampicillin. In order to make the GFP gene a functional one it has been engineered so the sugar arabinose triggers the production of the protein. The genes in the arabinoseoperon (araB, araA, and araD) have been replaced by the GFP gene. Such genes encode proteins that break down arabinose when it is present in the environment, so they are expressed only if this is the case. The regulatory sequence has been left intact, so in the engineered operon the presence of arabinose turns on the GFP gene and, therefore, GFP is produced.
  • Our results from using the pGLO kit…you get transformed E. coli (which have been plated on petri dishes) with the pGLO gene allowing for green fluorescence with UV light.
  • Fluorescent tags are easy to detect protein markers that can be inserted into a living organism. Different fluorescent tags respond to certain excitation wavelengths of light.GFP, for example, is a widely used fluorescent protein in bio imaging, however, each year, new fluorescent proteins are being developed to improve bio imaging techniques.Cells pic – Image to your left is Human lymphocyte cell, called a Jurkat E6-1 Cell. In the middle is a Jurkat labeled with GFP. The cell on the right is a Jurkat labeled with DsRed-Monomer (which is the current red fluorescent tag on the market).
  • We work with Thermosynechococcuselongatus or T. elongatus which has been isolated from the Beppu hot springs in Japan . You can think of this organism as “our jellyfish” where our goal is to modify its DNA , transform E. coli with the modified DNA, and finally, enable the transformed E. coli to fluoresce red. Our “big picture” goal is to have our red fluorescent tag be used in cellular imaging techniques in vitro and eventually in vivo, and as a teaching tool for bacterial transformation.
  • This picture shows current Infrared Fluorescent Proteins (IFPs). Our fluorescent protein is the one at the far right end of the graph called “SmuRF” which stands for small unexpected red fluorescent tag (another reason why it’s called SmuRF is because it looks blue to the eye when not under UV light!). Our fluorescent protein, compared to the others, has a red wavelength that will penetrate through tissues farther than the green and DsRed currently on the market. Kristen will talk about other benefits of our protein later.
  • We use two plasmids in our project…one is pBAD which codes for our protein and the other is pPL-PCB which codes for 2 enzymes that make our chromophore out of E. coli heme. In addition to these plasmids, we also use L-arabinose which turns on the operon of the pBAD plasmid, and IPTG which turns on the operon of the pPlL-PCB plasmid. Lastly, we also give our plasmids Kanamycin and Ampicillin resistance (note on picture). Kristen and our other team members will go more into depth with this later.
  • These are results of a recent protein expression completed this summer. Here we can see that 569T and 899T, our non-mutated protein, does show a bright yellow color while our mutated protein, 569TM shows a bright blue color. If these pellets were exposed to red exciting light, we would expect to see our 569TM fluoresce red in longer wavelengths.
  • Problems with pZazz…-Only few colonies fluoresce-We’ve been varying the amounts of IPTG+L-ara used for our plates to see if some amount combinations work better than others. -While we’re doing this, we also make sure to use the truncated only and truncated mutated forms of our proteins to compare our progress and to improve expression of “pZAZZ” plasmids to get bright red colonies (UV light).-SO….going to test out combos + mini cultures from glycerol stocks (w/IPTG & L-ara) = spin down pellet
  • Transcript

    • 1. Bioengineering Red Fluorescent Tags fromThermosynechococcuselongatus
      Bacterial Transformation WithpZAZZ
      Stefanie Yoshizuka
      September 19, 2011
    • 2. WHAT IS BACTERIAL TRANSFORMATION WITH pGLO?
      Green Fluorescent Protein (GFP) gene from Aequoreavictoria
    • 3. How does pGLO work?
       BioRadpGLO Kit
      DNA plasmid vector 
    • 4.
    • 5. Our Project
      • Bioengineer a red fluorescent tag
      • 6. Easy-to-detect protein marker
      • 7. Respond to different wavelengths of light
      • 8. Reporter of protein expression
      Photographs made at CBST – UC Davis
    • 9. Use in our project
      • T. elongatus isolated from hot springs in Beppu, Japan
      • 10. Cellular imaging techniques in vitro and eventually in vivo
      • 11. Teaching tool for bacterial transformation
    • Why make a red fluorescent tag?
    • 12. New fluorescent tag from T. elongatus
      899TM
      569TM
    • 13. Plasmids used to express the truncated cyanobacteriochrome 569TM
      pBAD plasmid + 569TM insert
      pPL plasmid
      GAF domain (protein)
      Phycocyanobilin (PCB, chromophore)
      Produce:
    • 14. Pellets From Protein Expression
      Centrifugation results

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