The GFP Project Week 0: Welcome to BOSSLAB

Something is cooking at BOSSLAB!

The GFP project has begun at BOSSLAB.  Headed by the Chief Troublemaker of this blog (me), a group of people will be using BOSSLAB to play around with pGREEN, a plasmid that produces GFP.

The goal of this project is not only to get local people working hands-on with biotech, but to support the DIYBIO community by producing a model project, with documentation, that people can do.  Over the next month or so I will be cranking out all the information you need to safely create and modify genetically engineered organisms in an environment about as sterile and complicated as your kitchen.

The GFP project will have # components, as listed  below:

  • Transform pGREEN intoe. Coli MM294
  • Extract pGREEN from the transformants and verify the quality of the plasmid DNA
  • Extract GFP from the transformants for extra points
  • ????

This week was kind of like “Week 0” for the project in that I showed everyone how to pour plates, streak for individual colonies, and practice (some amount) of aseptic technique.  As I mentioned, one of the goals for me in leading this project is to show people DIYBIO to show people basic techniques.  To make sure I do a good job, I will be covering the techniques we use in the project in detail in separate posts.

Hopefully we will get to do the first item next weekend, at the twice-montly BOSSLAB meeting.  It will be nice to have something really going on during the meeting; maybe more people will get interested and join the project, allowing us to get some nicer things for the project.

After this project comes ????.  That means I don’t know if we will continue doing molecular biology, or switch to something like microbial diversity, or try to do some kind of Mendelian genetics project with plants.  There are a lot of really cool biological things to investigate, and although molecular biology and synthetic biology are my favorites, BOSSLAB is really open to anything.  If we continue in the molecular biology direction, we could try doing a restriction digest on the extracted plasmid and run it in a gel, or have part of it sequenced (find out what kind of GFP we are making!), or ligate it to a localization tag and see it expressed in only part of the cell (contingient on getting our fluorescence scope working).  Really, there are all kinds of possibilities for this!  But first, extraction and verification have to happen.

BLYES: BioLuminescent Yeast Estrogen Assay

It looks like I will be doing some research for The Public Laboratory, trying to develop a yeast estrogen assay, specifically with yeast (BioLuminescent Yeast Estrogen Screen: BLYES).  Yay!  The overall goal of the project is to develop a kit that can be shipped to someone so they can, at some undetermined level of accuracy, determine the estrogenicity of their water.

Estrogenicity is an important metric that is best tested in vivo.  It   There are several factors that make it very difficult to asses otherwise.  The first problem is that there are a lot of chemicals, ranging from Bisphenol-A, which is found in many plastics, to birth control pills that all have estrogenic effects.  The other problem is that the only way to test how “estrogenic” something is to test it under the conditions that would be found in the body.  This is because of the unique estrogen receptors and chemicals like Sex Hormone Binding Globulin (SHBG) that inactivate (some) estrogens that are found in different biological systems.

Flow of estrogenic compounds

Unfortunately, humans grow very slowly and there is a lot of legislation that makes it difficult to test on them, not to mention the ethical concerns.  Also, it is not one or two compounds, but thousands of chemicals (with new ones added frequently) that need to be tested.  The EPA is actually in charge of testing these things, and has two tiers.  The first tier tests if it has endocrine disrupting effects, meaning (for us) if it is estrogenic or not, and if it should move on to tier two testing.  The second tier tests how much of an effect it has at different levels.  You can read what the EPA has to say here.

BLYES seems like a good test, because tricky things like specific receptors, and blood levels of SHBG can be simulated.  BLYES is uses a genetically engineered strain of yeast.  It is S. cerevisiae, the same species as the yeast that people use to bake bread or brew beer.

YES assay system. Circles are rough sketches of the plasmid. Estrogen interacts with hER-α receptor coded by pSCW231-hER, which causes the translation and transcription of β-galactosidase, which produces a colormetric change in some substrates

BLYES is really the combination of two earlier systems, one for detecting estrogen, and one for making yeast express bacterial luciferin.  The original system was called the Yeast Estrogen Screen (YES).  It is based around two plasmids.  One contained hER-α, and is called pSCW231-hER.  I have not yet found out what pSCW231 is, but it seems that it causes hER-α to be expressed in the membrane.  The other plasmid is YRpE2, which seems to stand for “Yeast Reporter Plasmid ERE 2”, because it has two ERE elements and contains the CYC1-lacZ fusion gene.  This gene codes for β-galactosidase, which breaks down ONGP to into something yellow.  This system, when exposed to estrogen, causes the hER-α to turn on the ERE, producing β-galactosidase.  The actual assay is done after allowing the yeast to grow in the media.  The cells are exposed to ONPG, and the “yellowness” of the result is read with a spectrophotometer.

the Lux operon. LuxA and LuxB code for the luciferase enzyme, while LuxCDE code for the substrate and complex that recycle it

The other component of BLYES is bioluminescence, which comes from two plasmids.  The genes for bioluminescence were inserted into two pBEVY (plasmid for Bidirectional Expression Vectors in Yeast) plasmids.  These carry the genes luxAB on one plasmid, named pUTK407, and luxCDE and frp on another plasmid called pUTK404.  luxAB codes for the luciferase that reacts with the FMNH(2) to produce light, while the luxCDE reduces the FMNH(2) so that it can be recycled to create more light.  frp codes for the FMNH(2).  The luxCDE plasmid is constitutively on because of its promoters, (GPD and ADH1).  In pUTK404, the genes frp, and Lux genes C, D, and E have Internal Ribosome Entry Sites (IRES).  These IRES ensure that the genes are properly transcribed by the ribosomes, instead of the ribosomes creating fusion protiens of LuxCD and LuxE-FRP.

BLYES: hER-α receptor is encoded by chromosomal DNA. Activation of the receptor turns on production of luxA and luxB under the control of bidirectional EREs. This enzyme reacts with the FRP produced by the constitutively on pUTK404 plasmid. pUTK404 also encodes LuxCDE, which allows the recycling of FRP. frp, and Lux genes CDE have IRES (internal ribosome entry sites) in-between them to ensure proper transcription

The difference is that BLYES has the human Estrogen Receptor alpha (hER-α) integrated into its chromosomal DNA.  This means that it is inside the nucleus.  Additionally, it is under a constitutive promoter.  The when it binds to an estrogenic chemical, hER-α turns on a signaling pathway that activates the Estrogen Response Element (ERE) promoter.  This promoter is on a plasmid that turns on light production with lux genes (bacterial luciferin/luciferase), and this is measured by a very sensitive detector.

Since The Public Laboratory wants to make this into a kit that they can send out to people, BLYES or YES may not be a good option, because they take very sensitive tools and extra chemicals to test estrogenicity.  Towards making this kit a possibility, we may try to link ERE activation to pigment production.

Transformation 3.0: Today I learned about Ampicillin

Today I learned that you should immediately plate bacteria that have ampicillin, and that it is SOP to do so in our lab here at Olin.  It doesn’t matter that you add them immediately, because they have time to inactivate the amp before they divide, which is when the amp kills them.  Maybe this explains all the satellite colonies I saw before!  I was waiting a good 15-30 minutes, which allows the bacteria to express beta-lactamase, which breaks down ampicillin.  This can destroy nearby ampicillin on the surface of plates, destroying any antibiotic effect, creating satellite colonies.  Oops.  Time to revisit my transformation protocol.