Took two weeks to get an appointment with Dr. lewis but was able to and it was extremely helpful. She explained that I was looking at the biolumenescence reaction wrong or not fullly. I was looking to effect the main reaction but had the genetic information for luceferase. luceferase is likely produced consistently and stored. by manipulating the oxygen levels it would effect the initial reaction but would likely have no effect on the animals producing luceferase. So when it went to check if levels of luceferase had been manipulating it by changing the amount of oxygen it may effect the initial reaction and change the amount of light produced but since oxygen is not related to the production of luceferase when i checked genetic information it is likely their would be not change.
oxygen depletion and ocean acidification manipulation have many other effects on the animals and it would be hard to tell if that is actually the cause of the change in the luminescence or it is just causing extra stress and not directly effecting the animal. Dr. Al-noori brought up in class that and easy way to effect the reaction would be to effect the inter-cellular calcium storage where the calcium used to initiate the release of luceferase from storage would be. using a calcium kelator, or inter-cellular calcium channel blocker may be more efficient since it would not allow the release of the luceferase
One thing to look at may be to look into checking if luceferase is indeed constantly produced or if it is only produced when needed. it is likely since the animal uses this as a defensive function that it would make sure that the levels were never depleted by constantly producing luceferase. Doing a MRNA-CDNA-PCR on incoming specimens and then another after light production may answer this question and clear up any questions. Dr Lewis mentioned that in order the truly understand the entire genes responsible for luminescence I would need to look at the entire genome by using a microaray. i will need to find out more information it may be beneficial to locate the genome since I do not believe it has been done as of yet.
The enclosure is complete setup and put together as well as the artemia enclosure. I placed 7 2 gallon buckets in the bottom with sifted find sand donated by the fisheries department. A timer with light has been setup but will need to be worked with to make sure it is on the correct light cycle.
Greg will be acquiring up the animals this coming Saturday 8/9/2014 from Bainbridge island Crystal Springs pier were I will pick them up after the dive and transfer them to uw
Have not been able to complete history portion of the proposal since I have had to many questions on the process of the experiment and do not feel confident enough to finish it but now that I was able to talk to Dr. Lewis, and Al-noori i think I have a clearer idea on what to do.
For the manipulation experiment I am going to put the animals in both and oxygen depletion, calcium manipulation and ocean acidification environment and then measure the illuminations with a camera to see if their is any change. This will also give me the ability to check the genetics to see if the production of luceferase is being manipulated. or if I am able to get the entire genome I may be able to look at more of the reaction and see what all genes are responsible for the process. I will talk to Dr. Roberts to look into it.
I also saw some information that states that the sea pens may illuminate at night so I would like to find a way to recorded the live stream and be able to look over it to see if this is true not only will it be wonderful to see but could give me the ability to test their luminescence without having to stress the animal out by touching it but will not know till they arrive and are comfortable in the setup.
i need to really comb through the reaction and pull as much information I am going to look into a permanent dry erase board that I can have access to to write out the entire reaction and be able to study and develop. It seems to help me understand better when I can step back and look and the entire thing. The quarter is coming to and end so I will need to continue the research next quarter which will give me the chance to do all of my experiments and know the pens are being taken care of. Building access was figured out so I can come in and take care of the pens even on weekends.
