Discovery Talk Questions and Answers: Developing GFP as a Biological Marker

Martin Chalfie

Questions

In this study, Dr. Chalfie and his team were able to demonstrate that heterologous GFP expression and activity did not require any additional gene products from A. victoria.
1. Why was this demonstration so important to the field of biology?
2. Describe the evidence provided by Dr. Chalfie and his team that GFP expressed in E. coli had the same activity as the GFP expressed in A. victoria.
List three key properties of GFP presented in this paper that make it an excellent candidate for “live” monitoring of cellular processes.
What additional property makes GFP an excellent candidate for neural cell monitoring?
Do you agree or disagree with the following statement?”The GFP produced in E. coli by Chalfie et al. has the same amino acid sequence as the endogenous GFP found in A. victoria.”
Explain why you agree or disagree, and provide evidence to support your statement.
Name two minor experimental steps that have been shown by Dr. Chalfie and his team to interfere with GFP activity.
In his talk, Dr. Chalfie explains that, if he had used the EcoRI fragment of cDNA encoding GFP instead of the PCR-amplified fragment, his experiment would have failed.However, reference 8 of his paper states that: “Plasmid pGFP10.1 contains the EcoRI fragment encoding the GFP complementary DNA (cDNA) from λgfp10 (3) in pBS(+) (Stratagene). The fragment was obtained by amplification with the polymerase chain reaction (PCR) with primers flanking the Eco RI sites and subsequent digestion with Eco RI.”
Explain what Dr. Chalfie and his team did, and whether or not it aligns with his statement in the video.
Describe the type of unpublished information Dr. Chalfie asked his colleagues for authorization to add to his paper.
How long did it take Dr. Chalfie to get the initial idea of using GFP as a marker of gene expression into an actual publication in Science?

Answers

a. Thanks to the work performed by Dr. Chalfie and his team, GFP is now a widespread biological marker used any many species. Although it was clear from the start to Dr. Chalfie that this protein had the potential to become such a widespread marker, he and his team needed to express the protein in its active form in other organisms than A. victoria. In particular, other scientists thought that GFP may require A. victoria-specific converting enzymes to get to its final active form. The fact that Dr. Chalfie’s student was able to express GFP in E. coli demonstrated that there was no need for such enzymes, and that GFP could be expressed in its active form in any organism.Video (3:31): “And because I was working on a transparent animal, Caenorhabditis elegans, I was looking at gene expression, I suddenly realized that this protein, GFP, would make a terrific marker for our experiments.”
Video (04:46): “When she did these experiments, there was hanging over us a problem. And that problem was that we weren’t really sure that it was going to work because of what was known about the GFP molecule. By this time investigators had found that GFP had a rather unusual post-translational modification. The peptide backbone in GFP cyclized, and the making of this five-membered ring was real mystery. People speculated that it might take one, two, or maybe even more converting enzymes to make the mature protein from the translated product. So it wasn’t a sure thing that this was going to work.”

p. 803: “Because this fluorescence requires no additional gene products from A. victoria, chromophore formation is not species-specific and occurs either through the use of ubiquitous cellular components or by autocatalysis.”

b. Figure 2 in the paper shows that E. coli-purified GFP has the same excitation and emission spectra as the endogenous GFP purified from A. victoria, and therefore the same activity as the original protein.
p. 803: “When GFP was partially purified from this strain (10), it was found to have fluorescence excitation and emission spectra indistinguishable from those of the purified native protein (Fig. 2). The spectral properties of the recombinant GFP suggest that the chromophore can form in the absences of other A. victoria product”
(1) Its detection does not require any additional factors, so tissues can be kept alive during monitoring
(2) Its detection requires UV or blue light, and therefore, the full signal can be detected without the limitation of substrates in the detection reaction
(3) It does not interfere with cell processes such as growth, so it can be used in live cells
p. 803: “Several methods are available to monitor gene activity and protein distribution within cells. These include the formation of fusion proteins with coding sequences for beta-galactosidase, firefly luciferase, and bacterial luciferase. Because such methods require exogenously added substrates and co-factors, they are of limited use with living tissue. Because the detection of intracellular GFP requires only irradiation by near UV or blue light, it is not limited by the availability of substrates. Thus, it should provide and excellent means for monitoring gene expression and protein localization in living cells. Because it does not appear to interfere with cell growth and function, GFP should also be a convenient indicator of transformation and one that could allow cells to be separated with fluorescence-activated cell sorting.”
The size of GFP is expected to help with monitoring proteins in small compartments or cellular regions, like is the case in neurons.
p. 803: “The relatively small size of the protein may facilitate its diffusion throughout the cytoplasm of extensively branched cells like neurons and glia.”
Disagree. The sequence used in the study published in this Science paper actually had an amino acid substitution in its final sequence.
p. 804, Section 8: “The sequence of the cDNA in pGFP10.1 differs from the published sequence by a change in codon 80 within the coding sequence from CAG to CGG, a change that replaces a glutamine residue with arginine. This replacement had no detectable effect on the spectral properties of the protein (Fig. 2)”
The authors found that nail polish (often used in histology techniques) and some anesthetics interfered with GFP fluorescence in C. elegans.p. 804, Section 9: “However, the chemicals in nail polish, which is often used to seal cover slips, did appear to interfere with the C. elegans GFP fluorescence.”
p. 804, Section 11: “Another nematode anesthetic, phenoxypropanol, quenched the fluorescence”
Dr. Chalfie and his team obtained the fragment described above but subsequently cloned it into the pET3 vector. To do this, they had to generate a new NHeI restriction site in the sequence and therefore produced an additional PCR fragment from the EcoRI sequence they had obtained.
p. 804, Section 8: “An E.coli expression construct was made with PCR to generate a fragment with an NheI site at the start of translation and an EcoRI site 5′ to the termination signal of the GFP coding sequence from pGFP10.1. (…) The NheI-EcoRI fragment was ligated into the similarly cut vector pET3a.”
Like in the talk, Dr. Chalfie mentions in the paper that some of the other investigators had issues producing a fluorescent protein from the EcoRI product. He suggest that thus may be because some 5’ elements in the sequence inhibit expression of gfp.p. 804, Section 8: “A similar PCR-generated fragment (1) was used in our C. elegans construct. As others are beginning to use pGFP10.1, we have heard that although similar PCR fragments produce a fluorescent product in other organisms (R.Heim, S. Emr, R. Tsien, personal communication; S. Wang and T.Hazelrig, personal communication; L. Lanini and F. McKeon, personal communication) (23), the EcoRI fragment does not (R.Heim, S. Emr, R. Tsien, personal communication; A. Coxon, J.R.Chailet, T.Bestor, personal communication). These results may indicate that elements at the 5′ end of the sequence or at the start of translation inhibit expression.”
Dr. Chalfie had the initial idea of using GFP as a marker on April 25, 1989 when Paul Brehm gave his talk about GFP. The paper was published on February 11th, 1994. It took about 5 years for Dr. Chalfie to turn his idea into paper.
Video (2:14): “Then on April 25th, 1989, I heard a seminar that really changed my life. It was a talk being given by Paul Brehm, who at the time, was a neurobiologist at Tufts University. And in the introduction of his talk, he started talking about the work of this man, Osamu Shimomura, and the work he had done in isolating proteins from the jellyfish, Aequorea victoria.”