Team:Valencia/other microalgae

From 2012.igem.org

Other Microalgae

Our aims go beyond building the most efficient photosynthetic biolamp, into trying to develop more robust and simple designs based on bioluminescent vascular plants. These kind of biolamps would be less energy-efficient, due to the lower surface-volume quotient and amount of structural carbon, but would be much easier to transport, manage, upkeep and commercialize. Vascular plants are robust organisms with a wider tolerance to changes in environmental conditions and independent of a broth vessel and complex tube systems (as they have their own biological structures for this function - xylems and phloems).

In first place, we would test of luminescence of S. elongatus WT with psbA-luxAB as grown broth biolamp, to assess the magnitude of the problem of pigment shading of luminescence.

The next step would be the transformation of Chlamydomonas reinhardtii's chloroplast with the lux cassette under ligth regulation .

Chlamydomonas reinhardtii:

Chlamydomonas reinhardtii is haploid unicellular green algae. It is about 10 micrometers long and 3 wide and has two flagella. Much of its volume is occupied by its only chloroplast which has 80 copies of its genome. Chlamydomonas can grow by autotrophic or heterotrophic ways using organic carbon and chemical energy sources.

This alga is widely spread around the world and is one of mostly used laboratory organism. It is used as model in studies of cellular movement, environmental stimuli responses and photosynthetic and cellular recognition studies.

Figure 1: Chlamydomonas reinhardtii world distribution. Source: http://www.algaebase.org/

In the industry this algae has been used on different areas such as: biofuels production (lipid for biodiesel), biorremediation (heavy metals subtraction from water), pigment production (carotenoids) and bioreactor for production of recombinant proteins for medical use. This is due to some of its features as easy and cheap culture, great capacity of store biomass and because commonly it is considered non harmful for human beings.

Table 1: Features comparation between different traditional organism used as bioreactors. Source: http://www.revistaciencia.amc.edu.mx/

Organisms	Production cost	Production time	Staggering capacity	Quality of product	Glucosilation capacity	Contamination risk	Storage cost
Bacteria	Low	Short	High	Low	No	Endotoxins	Moderated
Yeast	Medium	Medium	High	Medium	Yes	Low	Moderated
Insect cells	High	Medium	Medium	High	Yes	High	Expensive
Mamals cells	High	Long	Very low	Very High	Yes	Viruses prions	Expensive
Animals	High	Very long	Low	Very high	Yes	Viruses prions	Expensive
Plant cells	Low	Short	High	High	Yes	Low	Low cost
Plants	Very low	Long	Very high	High	Nucleus: yes Chloroplast: no	Low	Low cost
Microalgae	Very low	Short	Very long	High	Nuclus: yes Chlorplast: no	Low	Low cost

Experimental design:

Genetic circuit:

The designed genetic circuit is very similar to the one that is employed at co-culture project. In this one the psbA promoter regulates λ production; and this production of λ regulates the expression of lux operon.

During the day the presence of CI repressor inhibits the expression of lux operon because alga does not emit light.

Figure 2: Genetic circuit working in presence of light/during the day

During night λ product is not present and that allows the expression of lux operon and the following light emission.

Figure 3: Genetic circuit working in abscence of light/ during the night

Transform Chlamydomonas reinhardtii:

The psbAI gen is a chloroplast's gen. The reasons why we chose to transform chloroplast genome of Chlamydomonas reinhardtii are exposed in the next table:

Table 2: Comparison of nucleus manipulation against chloroplast manipulation

Strain selected:

With the intention of avoiding the use of antibiotics at selection of transformed cells we decided to employ the photosynthesis-deficient mutant strain ac-uc-221 (CC373) of C. reinhardtiii.

The ac-uc-2-21 is a deletion mutation in the chloroplast atpB gen, which results in a clean, non-reverting acetate-requiring phenotype. This strain was originally obtained from the culture collection of the Chlamydomonas Genetics Center at Duke University, NC, USA

The use mutant strain CC373 enables the selection of transformed cells employing vectors which carry the DNA of interest between plastome homologous regions and atpB gen. The atpB gen allows the restoration of a complete atpB gen copy. Then transformed cells will be able to grow in a culture medium without acetate and high light conditions while not transformed will not do.

Vector selected:

To introduce our construction and complement the deletion mutation ac-uc-2-21 we chose to use plasmid pCrc +157 (Inger Lill Anthonisen, 2002). This plasmid has between its has between inverted repeats homologous to plastome, regions XhoI and XbaI restriction sites flanking 5' rbcL (el promotor), uidA gen and 3' UTR from psaB (to stabilize transcripts).Apart it has atpB gen.

This plasmid has between its two regions with inverted repeat sequences homologous to plastome, targets for restriction enzymes Xhol and Xbal. Between these targets there is the uidA gen flanked by the rbcL 5' UTR and the psaB 3' UTR (this last stabilizes the transcripts). Out of these sequences it has the atpB gen.

Figure 4: expression cassette of pCrc+157. Source: Maria L. Salvador.

Method selected:

Chlamydomonas´ chloroplast can be transformed by different ways. The most prefered are Polyethilenglicol and gen gun methods. We chose gun gen because we found more literature explaining it. This consists on adhering your construction to gold or tungsten particles. After that you will shot them to your algae culture (in agar plates) at empty conditions.

Bullets will: - Fall and not hit algae. - Kill algae. - Break through algae. - Hit the chloroplast and transform it. As with all chloroplast transformation methods the introduction of desired DNA will be done by homologous recombination.

Future

This opens the way for further research on eukaryotic chloroplast transformation that can lead to the development of glowing trees on boulevards (see Team Cambridge 2011).

The last experimental step would be the transformation of Tobacco plant cell with psbA-lux cassette and development of a protoplast. The protoplast can be grown into a callus, from which we can finally obtain a bioluminescent plant.

References

Anthonisen I. L., Kasai S., Kato K., Salvadlor M. L., Klein U. (2002) Structural and functional characterization of a transcription-enhancing sequence elñement in the rbcL gene of the Chlamydomonas chloroplast genome. TRENDS in Plant Sciencie Vol. 7 Nº2
Blowers,A.D., Klein, U., Ellmore, G.S. and Bogorad,L (1993) Functional in vivo analyses of the 3' flanking sequences of the Chlamydomonas chloroplast rbcL and psaB genes. Mol. Gen. Genet., 238, 339-349.
Daniell H., Khan M.S. and Allison L. (2002) Milestones in chloroplast genetic engineering: an environmentally friendly era in iotechnology. Curr. Genet., 41: 349-356.
Maliga P. (2004) Plastid Transformation in Higher Plants. Annu.Rev. Plant Biol., 55:289-313.
Maria L.S., Loreto S. and Uwe K. (2011) Messeger RNA degradation is initiated at the 5' end and follows sequence - and condition-dependent modes in chloroplasts. Nuclei acids Research, 14, 6213-6222