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Protocols

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Project Participants

Ellen Widdup, Chelsea Leichman, Emily Hicks

July 8-14

Author: Chelsea, Ellen, Emily

Ellen, Chelsea, and Emily met with Dr. Alcantara (Joenel) and discussed achievements made thus far on the microalgae project (successful GUS transformation of Dt, outline of potential construct to be made in agrobacterium vector) and delegated specific tasks to members in the group for the next two weeks. Ellen and Chelsea plan to isolate nuclear and chloroplastic genomic DNA of Dt and potentially Chlamydomonas- this involves settling on a protocol and making sure all reagents/equipment are in order. Emily was assigned the task of building a vector for the agro construct.

Emily finalized and ordered primers for a microalgae construct. This construct includes a methyl benzoate-sensitive promoter and a luciferase reporter gene, and will be characterized in algae as a model system. Emily also made a list of important genes in microalgae to investigate for the purposes of biobricking.

July 15-22

Author: Chelsea, Ellen

This week we obtained a culture of D.t. from the Chua lab. We attempted to isolate genomic DNA from the culture. We have been trouble-shooting and adjusting our exisitng protocol (originally created for Arabidopsis) to try to obtain the DNA. So far we have had limited success but we are attempting some more trials. Tomorrow we will know if we have been succesful. A protocol will be provided once we determine the best method. In addition, we have obtained an effective protocol for isolating chloroplast DNA from Dr. Muench. Next week we should be able to isolate the chloroplast DNA from D.t. as well as obtain a Chlamydomonas culture from which to also isolate DNA.

On July 21st, Joenell Alcantara, a professor at the University of Calgary, told us that there was a published protocol for isolating DNA already, since the genome was already sequenced. Results next week.

July 23-28, 2011

Author: Chelsea, Ellen, Emily

This week we ran a gel of our isolated DNA to determine if our extraction protocol was successful. We got the correct sized band indicating that we do indeed have DNA. Since we have successfully isolated total DNA from the alga D.t. we can soon perform this for Chlamydomas (the model alga). We have been attempting to find a chloroplast DNA isolation protocol that will work. This DNA is needed to make the promoter library. So far we lack the equipment and/or expertise to undertake this kind of experiment. Despite this, we still plan to isolate chloroplast DNA from D.t. next week.

Emily tried amplifying microalgae luciferase for construction of a methyl benzoate reporter construct. Attempted to amplify other components of the construct, however further optimization is still needed. Next week, these will be attempted again.

July 29-August 4, 2011

Author: Chelsea

Objective

To isolate chloroplasts from Dunaliella tertiolecta

Summary

Isolated chloroplasts from Dt.

Reference

Holwerda, B.C., S. Jana, W.L. Crosby, Chloroplast and mitochondrial DNA variation in Hordeum vulgare and Hordeum spontaneum, Genetics 114, 1271-1291 (1986).

Protocol (adapted from reference)

1. Obtained subculture of Dt (~50 mL) and spun down (500 x g, 10 min, 25?C) to pellet cells out of culture media using swinging-bucket rotor.
2. Resuspended cells in 2 mL of isolation buffer.
3. Added ~.1 g 100um glass beads. Grinded mixture with mortar and pestle to break open cells on ice.
4. Pipetted mixture into a 2 mL eppendorf tube on ice. Let sit for 5 min. Pelleted out glass beads by centrifugation (200 rpm, 1 min).
5. Passed mixture through 2 layers of miracloth.
6. Centrifuged homogenate twice (1500 x g, 6 min, 4?C) in a swinging-bucket rotor.
7. Chloroplast pellets were resuspended in 16 uL resuspension buffer (15 mL sterile falcon tube)

Improvements

• grind pellet with sand and mortar and pestle before adding isolation buffer
• had issues with centrifugation at step 4- many cells appeared to be intermingled with the glass beads, hopefully the previous improvement would help remedy this
• miracloth may not be the best for small volumes- at least 1 mL absorbed by miracloth (even after squeezed)

Reagents

Isolation buffer – 50 mM Tris-HCl (pH 8), 25 mM EDTA, 10 mM 2-mercaptoethanol, 1.25 M NaCl, 0.1% BSA, Resuspension buffer – 50 mM Tris-HCl (pH 8), 25 mM EDTA

Interpretation

Although we got a pellet at the end of the isolation, we are unsure of a way to verify the purity of our chloroplasts (i.e. is there genomic DNA contamination?), and are not sure of the success of the isolation at this time. We intend to find this out ASAP. Further, the yield was quite low and we would likely repeat the experiment with the improvements suggested above before we isolate chloroplastic DNA.

August 5-12, 2011

Author: Chelsea and Ellen

Objective:

Grow enough Dt to isolate a significant yield of chloroplasts using the protocol from last week.

Summary:

Subcultured Dt so that we have ~2L of culture by next week to re-attempt chloroplast isolation and get a more significant yield.

August 13-19, 2011

Chelsea and Ellen

Objective:

Grow enough Dunaliella tertiolecta (Dt) to isolate a significant yield of chloroplasts using the protocol from two weeks ago. Our subculture last week did not grow well enough to provide us with enough Dt.

Summary:

We made culture media (3L) for Dt, consisting of:

• 60 mL Guillard’s (f/2) marine water enrichment solution 50x
• 1.5 L artificial sea water
• 1.5 L ddwater

The media was mixed, pH’d to 8.0, aliquoted and autoclaved. Next, we subcultured Dt again so that we will have ~2L of culture by next week to re-attempt chloroplast isolation and get a more significant yield.