Title: Bioengineering Small, Bright Red Fluorescent Reporters from Cyanobacteria T. Elongatus BP-1

Outcome: The focus of this research project is to bioengineer a small, bright red fluorescent reporter from the cyanobacterium (a blue-green bacteria that obtains its energy through photosynthesis), Thermosynechococcus elongatus BP-1. The endpoint of this research is to insert this red reporter into the cytoskeleton (the network of internal cellular fibers) of a mammalian Jurkat cell (Figure 1b). The Jurkat cell is a white blood cell targeted by HIV infection. By successfully tagging the cytoskeleton with the red reporter one is able to visualize the internal network of microfibers that draw the infectious particle into the nucleus of the cell. In the test system the red fluorescent Jurkat cytoskeleton is distinguishable from green fluorescent HIV viral particles (Figure 1a). This will allow researchers to monitor the movement of HIV into living cells during infection. A greater understanding of the infection process between red-tagged Jurkat cells’ cytoskeleton and green-tagged HIV will allow scientists to develop more effective tools to combat infection.

The Research: The Spiller Research group engineered a truncated mutant (called 569TM) of T. elongatus cyanobacteriochrome that fluoresces at 680nm, red wavelengths (Figure 2).  This discovery suggests that 569TM is a viable candidate as a small, bright red fluorescent reporter for tagging the proteins of the cytoskeleton of the Jurkat cell system. The next step will be to insert the red fluorescent tag into the cells shown in Figure 1b. These cells presently contain DsRed-Monomer Actin plasmid that causes their cytoskeleton to glow orange-red. The novel plasmid would cause the cells to make red fluorescent 569TM-Actin. The unique contribution of the tag will be fluorescence at a longer red wavelength for better visualization deeper into the cell. In addition, we think that the tag will have fewer side effects that could damage the cell than the DsRed-Monomer tag.

Figure 1a: Green-tagged HIV system infecting a Jurkat lymphocyte cell. Figure 1b: DsRed-Monomer Actin tagged Jurkat lymphocyte cell cytoskeleton. The marketed DsRed-Monomer reporter will be replaced by the bioengineered 569TM reporter.Credit for Figure 1a: Wolfgang Hübner et. al, Quantitative 3D Video Microscopy of HIV Transfer Across T Cell Virological Synapses, Science 323, 1743-1747 (2009). Credit for Figure 1b: Susan Spiller, Ph. D (spiller@mills.edu); Mills College-California Deanna Thompson, graduate student CBST.

Figure 2: Purified protein sample of 569TM. This small protein fluoresces bright red at 680nm suggesting 569TM is a better reporter candidate than DsRed-Monomer, which has a fluorescence maximum at 586 nm (orange-red).Credit for Figure 2: D. Brenda Salantes (dsalantes@gmail.com); Mills College-California

Impact/benefits: Research in biophotonics at Mills College has made novel contributions to the scientific community. The educational outreach program at Mills is supported by the Center for Biophotonics Science and Technology (CBST) and NSF CBET 0967965. This collaboration additionally allows undergraduates and recent Mills graduates to meet academic scientists at University of California Davis. In turn the Mills students reach out to students and teachers at a local high school for the advancement of science. By exposing high school students to research in biophotonics, Mills students encourage them to enter areas of biomedical science. The students are able to learn about the progress of ongoing research during the academic year in addition to developing their own scientific inquiries, which are presented at a competitive science symposium at UC Davis, CBST.

Background/Explanation: Currently there are several infrared fluorescent proteins on the market that are being used as fluorescent reporters. However, these tags have limited imaging properties, are longer in amino acid length, susceptible to photo bleaching, and release a reactive oxygen molecule that causes chemical damage to the cell. Improved fluorescent reporters can be bioengineered from cyanobacteriochromes from the cyanobacterium T. elongatus. We have currently shown that the bioengineered reporter, 569TM of T. elongatus, is shorter in amino acid length (making it a less invasive reporter), is less susceptible to photo bleaching, and will not cause oxidative chemical damage to the cell.

In the past, there have been no known protocols that were successful in bioengineering small, bright red fluorescent reporters from T. elongatus.  The usefulness of this proposed reporter has been recognized by CBST UC Davis, CBST Mills College and the NSF. However, before we can proceed with implementing our reporter into a mammalian cell system we must develop a protocol that translates our prokaryotic T. elongatus sequence into a DNA sequence that is compatible within a mammalian cell. Mills CBST has been successful in tagging the cytoskeleton of

Jurkat cells with marketed red fluorescent reporter DsRed-Monomer Actin. Future work will include replacing DsRed-Monomer of the Actin gene with translated 569TM sequence.

Figure 3a Expressed proteins in E.coli of T. elongatus reporter candidates. Bright blue pellet on the right indicates successful expression of mutated protein, 569TM. Figure 3b:  Students of EOSA (East Oakland School of the Arts) watch as their UV painted cells fluoresce under a UV lamp. This classroom activity led by Mills CBST teaches students the usefulness of fluorescent reporters in visualizing a cell.Credit for Figure3a and 3b: D. Brenda Salantes (dsalantes@gmail.com); Mills College-California

Directorate/Division: Engineering (ENG) / Chemical, Bioengineering, Environmental, and Transport Systems (CBET).

Program Officer: Leon Esterowitz

NSF Award Number(s): CBET 0967965

Award Title: Development of Bioengineered Cyanobacteriochromes for in vivo Fluorescence Tagging

PI Name(s): Susan Spiller, Ph.D.

Institution Name(s): Mills College