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Experiments with Thalassiosira weissflogii (TW) in Relation to Changing Oceanic Carbon Dioxide Concentrations. Raheel Anwar Morel Lab. CO 2 Concentrations and Why They’re Important.

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Raheel anwar morel lab

Experiments with Thalassiosira weissflogii (TW) in Relation to Changing Oceanic Carbon Dioxide Concentrations

Raheel Anwar

MorelLab


Co 2 concentrations and why they re important

CO2 Concentrations and WhyThey’reImportant

  • Thechangingenvironment has ledtohigherdissolvedcabrondioxide concentrations in theworld’soceansdueto global warming and thegreenhouse gas effect

  • Higher CO2 concentrations,which can easilybeconvertedtobicarobonate, cause changes in the pH oracidity of theworld’soceans


Raheel anwar morel lab

http://switchboard.nrdc.org/blogs/lsuatoni/why_scientists_agree_ocean_aci.html


The cdca enzyme

The CDCA Enzyme

  • Cadmiumcarbonicanhydrase

    • Catalyzestheconversion of CO2 intobicarbonate and viceversa

    • Cadmiumis a normallytoxic metal, buthere, it can act as a coenzymeforthereaction


Why tw and cdca

Why TW and CDCA?

  • Knowing where and in what quanitities thisenzymeislocated in the algae is important

    • Tells ushowthisspecies and otheralgaelikeit can cope with the changing conditions of the oceans

    • Allow ustoanticipate the effects of future changes


Overview of work

Overview of Work

  • Studiedtheexpression of the CDCA enzyme in TW underdifferent pH conditions

  • Isolatedchloroplaststo determine CDCA activity and localization


Cdca analysis

CDCA Analysis

  • Looked at TW cells growing under:

    • High (8.6)

    • Medium (8.2) and

    • Low (7.8) pH conditions.


Western blot

Western Blot


Isolation of chloroplasts

Isolation of Chloroplasts

  • Developed a procedurefortheisolation of chloroplasts

  • First, sonicationwasusedto break open cellswithoutentirelydestroyingallthechloroplasts

    • Cells are resuspended in a buffer designedtoprotectthechloroplaststhat are nowfloating free in thesolution

    • Solutionisfilteredon a 10 micronfilter, allowing TW cells, most of which are 10 micronorbiggertobecaughtonthefilter, whilechloroplasts, whichnecessarily are smaller, passthrough.

    • This reduces wholecellcontaminants remarkably

    • Chloroplasts are thenisolatedon a gradientforfurtheranalysis


Mims assay of cdca activity for chloroplast extract

MIMS Assay of CDCA activity for Chloroplast Extract

  • Results showed that CDCA activity was not present in the chloroplasts

    • Reasons:

      • CDCA enzyme could be located outside of the chloroplasts

      • Whole cell activity is ruled out since the pellet of chloroplasts is washed many times

      • Chloroplasts could get inactivated, but fluroescence microscopy shows only slight decrease in output

      • Signals from the nucleus may be necessary for CA activity


Conclusions

Conclusions

  • Overall, higherconcentrations of CDCA are foundwhenthe pH ishigher, givinguscluesonhowalgae in theoceansare effected

  • Isolation of chloroplastsshowed no signs of CDCA activity


Thanks to

Thanks to

  • PEI/Grand Challenges

  • Dr. Yan Xu

  • Professor Morel


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