12/22/2014

Objective: Have Sam run a gel using PCR product created from Sea Pen cDNA
PCR Gel
Gel run by sam

Final samples collected
1 daylight sample for 4 of the sea pens
1 dark sample for 4 of the pens

Final samples animals were euthanized by putting them into the -80C

Results:
e-mail:
"Unfortunately, your PCR didn't work, so there wasn't anything to cut out and purify. The issue this time is different than what you had before. In this instance, no distinct band was produced. For some reason, your sample is just a smear. The ladder run on the gel is the O'GeneRuler 100bp Ladder.
Looking at the size of the smear, and the fact that you have just a smear and no distinct band(s), suggests that what you have on the gel is RNA or straight cDNA. However, it's a LOT of RNA/cDNA (based on the intensity of the fluorescence)."


Inline image 2

10/7/2014
Objective: Verify Sea Pen luciferase sequence was sucessfuly replicated using primerse based on  Renilla Reniformes luciferase sequence acquired on Genebank.

PCR gel run today
Sea pen 1 and 2
1-1 lane 2 (note: Made sea pen luminescence before taking samples on both 1-1 (base cut) and 1-2 (top cut) )
1-2 lane 3 (note: Made sea pen luminescence before taking samples on both 1-1 1-2)
2-1 lane 4
2-2 lane 5
- lane 6
- lane 7
Bioline hyper ladder 1








Joined Dr. Roberts class lab and performed a conventional pcr (2x Apex Red)
6 tube 1-1,1-2,2-1,2-2,and 2 negative control tubes
master mix
2x apex red 12.5ul*6 =75ul
f primer 0.5ul*6 3ul

r primer 0.5ul*6 3ul
h20 6.5ul*6 39ul
=20ul per tube
template 5ul*6 30ul

95c-10 min
40 cycles of
95c 15s
50c 15s
72c 1min

Results: Replication was successful removed the brightest bands in lane 3 and 4 from the gel, and purified to prepare for sequencing. Samples were sent to "" (will add later)
 Found out that the problem with the reverse transcription quantities was a pipetting error on the 10ul pipet i was using to much because of a mix up on the decimal place of the pipette

Introduced a spacer for the sea pens to keep them from touching each other testing it on the 5 gallon bucket . It splits the bucket in half reducing the area they are able to move around in without blocking flow or height the pen can extend Will see if it improves individual health of the sea pens.

With the automatic feeder I have noticed that the Pens have become much more consistent in activity so I believe I am set it up so that all of them can be sustained for a long period of time though more time will be needed to tell if this is true. It may be beneficial to create a paper just on my setup which could help others working with sea pens or related species.

10/2

Objerctive: Convert RNA to cDNA using

Methods:

Reverse Transcription (Promega M-MLV: Cat#M1701; ) [Cost per sample ~$1.50]

A single reaction volume = 25uL. The volume of RNA, primer(s) and M-MLV RT used are variable and will be specific to your current experiment. The directions below apply to a reaction using 1ug of total RNA. You may need to make changes to accommodate your own conditions.

1. Use as much RNA as possible (up to 1ug); max volume of RNA = 17.75uL. Generally, identify the RNA sample with the lowest concentration and multiply by 17.75uL. Use this quantity (ug) of RNA for each and every sample.
2. Transfer calculated volume(s) of RNA to 0.5mL snap cap tubes or PCR plate. Adjust volumes of individual samples to 17.75uL with H2O.
3. Add appropriate amount of primer to sample. Use 0.25ug primer per 1ug of RNA in sample (= 0.5uL of Promega oligo dT Cat#C1101 in this example). Total volume (RNA + primers) should equal 18.25uL.
4. Heat samples at 70C for 5 min in thermocycler.
5. Place samples on ice IMMEDIATELY.
6. Make Master Mix:

PER RXN
5 uL 5x Buffer (M-MLV RT Buffer)
1.25 uL 10mM dNTPs (Promega Cat#U1511)
0.5 uL M-MLV RT per ug of RNA

7. Mix well.
8. Add 6.75uL of master mix to each reaction.
9. Mix well, but do not vortex.
10.Spot spin.
11.Incubate @ 42C for 1hr in thermalcycler for oligo dT primers OR @ 37C for random primers.
12.Heat inactivate @ 95C for 3 min.
13.Spot spin.
14.Store @ -20C.

3/17/2011 SJW

Starting RNA 1ug calculations
1-1 .99ul
1-2 2.31ul
2-1 1.13 ul
2-2 1.50 ul

Results:  Procedure was going well until we reached the Master Mix solution. noticed that their was to much master mix left over so it is likely somewhere in the creation of the MM or pipetting the MM into the solution that an error was made. We still ran the entire protocol to see if anything was successful. Planning on running the PCR next week to verify that the primers that were ordered are valid.

Checked on the Sea pens today the changes to the peristaltic pump appeared to be successful everything was running correctly and the levels in the feeding bucket were the same as when I left yesterday. I am still considering removing the reverse flow on the pump into the bucket so the quantities of artemia being delivered are more consistent but will watch the sea pens to see if this makes a difference. I noticed that in each of the buckets with two sea pens that 1 sea pen is very inflated and looks healthy and the other does not so it is possible that they do not do well when in close quarters with each other possible because they are attacking each other when touching. I would like to take some tissue samples to verify that they in fact do have nematocysts though the I will have to look into if it would be visible just by looking through the microscope may need to find a way to fire them off which we had done in lab I will talk with Claire to see.

10/1

Peristaltic pump began leaking and flooding aquarium room today after some investigation I found the hose being used for the pump was breaking down. I replaced the hose but the next day it wound up in the rotar and destroyed the hose. After talking with Jon about possible solutions I made changes to the pump by adding more rotars to help hold everything in place and added wing nuts to secure everything. Regular rotation of the tubing will also help to reduce breakdown of the tubing (note: ask greg about ordering some peristaltic tubing to reduce issues.

Redesigned the feeding schedule to match tide fluctuations. Currently setup to feed for 3 hours at 6am which mirrors the sea pens normal feeding regiment better than 6 times a day for an hour. it is likely the pens were not getting enough access to the artemia.

I would like to look into the feeding habits of the sea pen and sea if I can get data to support the sea pens using luminescnece to attract artemia.

Took samples from two of the sea pens removing pieces of fringes from the top and lower sections with scissors and a scalpol. Removed each of the pens from the water and placed on a tray. (notes: sea pens were very heavy and filled with water. once out of the water they started to leak the water reducing in size. With touch the pens started luminescing and started to shrivel making it difficult to take tissue from them. Best method was to quickly take tissue as soon as taken out of water so that the specimen did not have the chance to shrivel. One Pen was nicked on the inside of the stock and water rushed out. The Pens inflate themselves like a balloon increase greatly in volume in the water.) The sea pen recovered fully and it behavior in the tank stayed consistent with the others. it was able to inflate lower area below cut enabling it to grow not sure now it separates the area that was cut. Both pens seems to not be effected to harshly from the removal of tissue.

Sam went through everything for the RNA extraction going over step by step and teaching me the location and how to use all reagents and protocol. RNA extraction was a success numbers were successful

Made changes to the feeding schedule and setup.  added a paralytic pump with a line pushing artemia into the tank and another pulling water into the 5 gallon food storage bucket. feeding every 6 hours for 1 hour.

Helped Jake with counts at Taylor shellfish and with maintenance in Olympia cleaning trays doing counts and transferring them to new cages.

An interest in bioluminescence original idea that it is a way to attract a predator to what is eating it is unlikely since what eats sea pens is not preyed upon by many thing that use visual ques. Plus the idea that sea pens light scares off what ever is trying to eat it is not supported since nudibranches, and starfish due not rely heavily on vision. After talking with Jake, and Professor Jensen my thought is that since artemia and other zooplankton are attracted to light it would make sense that a sea pen would light up to attract its prey.

Ordered more artemia from greg which has yielded much more artemia with each batch the past eggs were very old and it was very hard to keep consistent with the amount of artemia.  When adding new artemia to the 5 gallon bucket 3 gallons are added and 1 gallon is added directly to the tank. The pump does not seem to be consistant with the amount of water it is moving back into the bucket so making some changes to equalize it may be nessecary though it is not enough yet to cause any issues. I had taken away the automatic feeder that was adding marine food flake to the aquarium and quickly noticed a decline in the sea pens. The note that they are detritivoreis supported I think they feed on both the artemia and the flake food that is made up of many diffrent types of marine ingredients.

Omega one Marine flakes:

Ingredients:
Whole Herring, Whole Salmon, Halibut, Black Cod, Seafood Mix (Krill, Rockfish, Shrimp, Squid, Clams, Salmon Eggs, and Octopus), Wheat Flour, Wheat Gluten, Fresh Kelp, Spirulina, Garlic, Lecithin, Astaxanthin, L-Ascorbyl-2-Phosphate (Source of Vitamin C), Natural and Artificial Colors, Vitamin A Acetate, Vitamin D3 Supplement, Vitamin E Supplement, Vitamin B12 Supplement, Riboflavin, Niacin, Pantothenic Acid, Folic Acid, Biotin, Inositol, Tocopherol (Preservative), Ethoxyquin (Preservative).
Guaranteed Analysis:
Crude protein (min) 43%, crude fat (min) 12%, crude fiber (max) 2%, moisture (max) 8.5%, ash (max) 8%, phosphorus (min) .5%, omega 3 (min) 2%, omega 6 (min) 1%.

9/25 First mortality occurred the upper portion (fringes) seemed to be disintegrating  and the upper structure became very stiff and hard though the lower section seemed to still be alive which may be since this a colonial organism if one part dies the other may still be able to survive though it is likely without the ability to gather food would perish very quickly. The process happened within 2 days change was fairly rapid. (Note: the sea pen was in close proximity to other sea pens I am wondering if it they may attack each other when to close. Speaking with Greg he noted that in the wild they are not found close to one another so when touching it is possible that they use some defense to attack the others possibly using feeding structures (autozooids) with nematocysts.

7/24-8/8

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. 

7/24

Had a meeting with Dr. Lewis from UW Bothell to help clarify some biology concepts about genetics and to help understand choosing primers and interpreting results of other researchers Genetic results. Choose primers so I can now order them for research.

Primer potentials

forward CGGCAAAAGCTAGCGTTGAA

reverse    ACGTGCGGTCTCTTTATGCT

7/21

Got in contact with Greg and he agreed to collect the specimens for me.

Artemia are looking like the best bet for feeding the pens, Talked with Greg today and he recommended it as well. Since the tank is connected to a larger system I do not want to throw off anything plus the artemia are easy to work with.

7/22-7/23

Finished preparing everything for the aquarium, setup power-heads with adapter that constantly rotates to create a more "chaotic" current through out tank that Pens prefer.

Sanitized and Setup artemia hatching enclosure with airline tubing connected in to air outlet spoke with Greg and he gets specimens we will be able to order in Artemia eggs, Will hatch them regularly so that the Pens will have a consistant food source.

Found DNA sequence for luciferase and potential primers looking into how to choose primers to fit correctly.

Added more to Proposal should have more time after midterms are finished tomorrow to finish it up and have it signed by Dr. Roberts.


http://www.ncbi.nlm.nih.gov/nuccore/HV097611.1

Ptilosarcus sp. CSG-2001 green fluorescent protein (GFP) mRNA, complete cds

Sequence ID: gb|AY015995.1|Length: 1094Number of Matches: 2

Related Information

Range 1: 18 to 764GenBankGraphics Next Match Previous Match

Alignment statistics for match #1

Score	Expect	Identities	Gaps	Strand
1369 bits(741)	0.0	745/747(99%)	0/747(0%)	Plus/Plus

Query  1    GACAAAATGAACCGCAACGTATTAAAGAACACTGGACTGAAAGAGATTATGTCGGCAAAA  60
            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct  18   GACAAAATGAACCGCAACGTATTAAAGAACACTGGACTGAAAGAGATTATGTCGGCAAAA  77

Query  61   GCTAGCGTTGAAGGAATCGTGAACAATCACGTTTTTTCCATGGAAGGATTTGGAAAAGGC  120
            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct  78   GCTAGCGTTGAAGGAATCGTGAACAATCACGTTTTTTCCATGGAAGGATTTGGAAAAGGC  137

Query  121  AATGTATTATTTGGAAACCAATTGATGCAAATCCGGGTTACAAAGGGAGGTCCGTTGCCA  180
            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct  138  AATGTATTATTTGGAAACCAATTGATGCAAATCCGGGTTACAAAGGGAGGTCCGTTGCCA  197

Query  181  TTCGCTTTCGACATTGTTTCCATAGCTTTCCAATACGGGAATCGCACTTTCACGAAATAC  240
            ||||||||||| ||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct  198  TTCGCTTTCGATATTGTTTCCATAGCTTTCCAATACGGGAATCGCACTTTCACGAAATAC  257

Query  241  CCAGACGACATTGCGGACTACTTTGTTCAATCATTTCCGGCTGGATTTTTCTACGAAAGA  300
            ||||||||||||||||||||||||||||||||||| ||||||||||||||||||||||||
Sbjct  258  CCAGACGACATTGCGGACTACTTTGTTCAATCATTCCCGGCTGGATTTTTCTACGAAAGA  317

Query  301  AATCTACGCTTTGAAGATGGCGCCATTGTTGACATTCGTTCAGATATAAGTTTAGAAGAT  360
            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct  318  AATCTACGCTTTGAAGATGGCGCCATTGTTGACATTCGTTCAGATATAAGTTTAGAAGAT  377

Query  361  GATAAGTTCCACTACAAAGTGGAGTATAGAGGCAACGGTTTCCCTAGTAACGGACCCGTG  420
            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct  378  GATAAGTTCCACTACAAAGTGGAGTATAGAGGCAACGGTTTCCCTAGTAACGGACCCGTG  437

Query  421  ATGCAAAAAGCCATCCTCGGCATGGAGCCATCGTTTGAGGTGGTCTACATGAACAGCGGC  480
            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct  438  ATGCAAAAAGCCATCCTCGGCATGGAGCCATCGTTTGAGGTGGTCTACATGAACAGCGGC  497

Query  481  GTTCTGGTGGGCGAAGTAGATCTCGTTTACAAACTCGAGTCAGGGAACTATTACTCGTGC  540
            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct  498  GTTCTGGTGGGCGAAGTAGATCTCGTTTACAAACTCGAGTCAGGGAACTATTACTCGTGC  557

Query  541  CACATGAAAACGTTTTACAGATCCAAAGGTGGAGTGAAAGAATTCCCGGAATATCACTTT  600
            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct  558  CACATGAAAACGTTTTACAGATCCAAAGGTGGAGTGAAAGAATTCCCGGAATATCACTTT  617

Query  601  ATCCATCATCGTCTGGAGAAAACCTACGTGGAAGAAGGAAGCTTCGTGGAACAACACGAG  660
            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct  618  ATCCATCATCGTCTGGAGAAAACCTACGTGGAAGAAGGAAGCTTCGTGGAACAACACGAG  677

Query  661  ACGGCCATTGCACAACTGACCACAATTGGAAAACCTCTGGGCTCCCTTCATGAATGGGTG  720
            ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct  678  ACGGCCATTGCACAACTGACCACAATTGGAAAACCTCTGGGCTCCCTTCATGAATGGGTG  737

Query  721  TAGAAAATGACCAATATACTGGGGAAA  747
            |||||||||||||||||||||||||||
Sbjct  738  TAGAAAATGACCAATATACTGGGGAAA  764

Range 2: 766 to 1094GenBankGraphics Next Match Previous Match First Match

Alignment statistics for match #2

Score	Expect	Identities	Gaps	Strand
344 bits(186)	1e-90	295/341(87%)	34/341(9%)	Plus/Plus

Query  941   CGATAACCGTTTGGAAGCTTGTGTATACAAGTTATTTGGGGTCATTTTGTAATGTGTATG  1000
             |||||||||||||||||||||||||||||| |||||||||||||||||||||||||||||
Sbjct  766   CGATAACCGTTTGGAAGCTTGTGTATACAAATTATTTGGGGTCATTTTGTAATGTGTATG  825

Query  1001  TGTGTTGTATGATCTATAGACGTCGTCATTCATAGCTTGAATCCTTCAGCAAAA------  1054
             |||||||||||||| |||||||||||||||||||||||||||||||||||||||      
Sbjct  826   TGTGTTGTATGATCAATAGACGTCGTCATTCATAGCTTGAATCCTTCAGCAAAAGAAACC  885

Query  1055  ------------GAAACCTCGAAGCATATTGAAACCTCGACGGAGAGCATAAAGAGACCG  1102
                         |||||||||||||||||||||||||||||||||||| |||||||||||
Sbjct  886   TCGAAGCATATTGAAACCTCGAAGCATATTGAAACCTCGACGGAGAGCGTAAAGAGACCG  945

Query  1103  CACGTAC-A-CAAATTATAATACCAGCAGTTGGAATCTTTAAACCGATCAAAACTATTAa  1160
             |||  |  | |   || | | |||||||||||||||||||||||||||||||||||||||
Sbjct  946   CACAAATTAACGCGTT-TCA-ACCAGCAGTTGGAATCTTTAAACCGATCAAAACTATTAA  1003

Query  1161  tat--atatatacaccctgtataacatatatatatatatatatCTACATAGTTTGATATT  1218
             |||  ||||||| |||||||||||| | ||||||||| |||      |||||||||||||
Sbjct  1004  TATAAATATATATACCCTGTATAAC-T-TATATATATCTAT------ATAGTTTGATATT  1055

Query  1219  GATTAAATCTGTTCTTGATCACTaaaaaaaaaaaaaaaaaa  1259
             |||||||||||||||||||||  ||||||||||||||||||
Sbjct  1056  GATTAAATCTGTTCTTGATCA--AAAAAAAAAAAAAAAAAA  1094

JP 2009148270-A/20: LUCIFERASES, FLUORESCENT PROTEINS, NUCLEIC ACIDS ENCODING THE LUCIFERASES AND FLUORESCENT PROTEINS AND THE USE THEREOF IN DIAGNOSTICS, HIGH THROUGHPUT SCREENING AND NOVELTY ITEMS

GenBank: HV097611.1

FASTA Graphics

Go to:

LOCUS       HV097611                1279 bp    DNA     linear   PAT 15-JUL-2011
DEFINITION  JP 2009148270-A/20: LUCIFERASES, FLUORESCENT PROTEINS, NUCLEIC
            ACIDS ENCODING THE LUCIFERASES AND FLUORESCENT PROTEINS AND THE USE
            THEREOF IN DIAGNOSTICS, HIGH THROUGHPUT SCREENING AND NOVELTY
            ITEMS.
ACCESSION   HV097611
VERSION     HV097611.1  GI:340198043
KEYWORDS    JP 2009148270-A/20.
SOURCE      Ptilosarcus gurneyi
  ORGANISM  Ptilosarcus gurneyi
            Eukaryota; Metazoa; Cnidaria; Anthozoa; Octocorallia; Pennatulacea;
            Subselliflorae; Pennatulidae; Ptilosarcus.
REFERENCE   1  (bases 1 to 1279)
  AUTHORS   Szent-gyorgyi,C. and Bryan,B.J.
  TITLE     LUCIFERASES, FLUORESCENT PROTEINS, NUCLEIC ACIDS ENCODING THE
            LUCIFERASES AND FLUORESCENT PROTEINS AND THE USE THEREOF IN
            DIAGNOSTICS, HIGH THROUGHPUT SCREENING AND NOVELTY ITEMS
  JOURNAL   Patent: JP 2009148270-A 20 09-JUL-2009;
            Bruce Bryan,Christopher Szent-Gyorgyi,PROLUME LTD
COMMENT     OS   Ptilosarcus gurneyi
            PN   JP 2009148270-A/20
            PD   09-JUL-2009
            PF   05-JAN-2009 JP 2009000303
            PR   01-OCT-1998 US    60/102939,27-MAR-1998 US    60/079624, PR
            15-JUN-1998 US    60/089367
            PI   christopher szent-gyorgyi,bruce j bryan
            CC   Ptilosarcus Green Fluorescent Protein (GFP) (insert B) FH
            Key             Location/Qualifiers
            FT   CDS             (7)..(720).
FEATURES             Location/Qualifiers
     source          1..1279
                     /organism="Ptilosarcus gurneyi"
                     /mol_type="unassigned DNA"
                     /db_xref="taxon:161601"
ORIGIN      
        1 gacaaaatga accgcaacgt attaaagaac actggactga aagagattat gtcggcaaaa
       61 gctagcgttg aaggaatcgt gaacaatcac gttttttcca tggaaggatt tggaaaaggc
      121 aatgtattat ttggaaacca attgatgcaa atccgggtta caaagggagg tccgttgcca
      181 ttcgctttcg acattgtttc catagctttc caatacggga atcgcacttt cacgaaatac
      241 ccagacgaca ttgcggacta ctttgttcaa tcatttccgg ctggattttt ctacgaaaga
      301 aatctacgct ttgaagatgg cgccattgtt gacattcgtt cagatataag tttagaagat
      361 gataagttcc actacaaagt ggagtataga ggcaacggtt tccctagtaa cggacccgtg
      421 atgcaaaaag ccatcctcgg catggagcca tcgtttgagg tggtctacat gaacagcggc
      481 gttctggtgg gcgaagtaga tctcgtttac aaactcgagt cagggaacta ttactcgtgc
      541 cacatgaaaa cgttttacag atccaaaggt ggagtgaaag aattcccgga atatcacttt
      601 atccatcatc gtctggagaa aacctacgtg gaagaaggaa gcttcgtgga acaacacgag
      661 acggccattg cacaactgac cacaattgga aaacctctgg gctcccttca tgaatgggtg
      721 tagaaaatga ccaatatact ggggaaaatc accaatatac tggggaaaat gaccaattta
      781 ctggggaaaa tgaccaatat actgtagaaa atcaccaata tactggggaa aatgaccaat
      841 ttactgggga aatgaccaat ttactgtaga aaatcaccaa tatactgtgg aaaatgacca
      901 aaatactgta gaaatgttca cactgggttg ataaccgttt cgataaccgt ttggaagctt
      961 gtgtatacaa gttatttggg gtcattttgt aatgtgtatg tgtgttgtat gatctataga
     1021 cgtcgtcatt catagcttga atccttcagc aaaagaaacc tcgaagcata ttgaaacctc
     1081 gacggagagc ataaagagac cgcacgtaca caaattataa taccagcagt tggaatcttt
     1141 aaaccgatca aaactattaa tatatatata caccctgtat aacatatata tatatatata
     1201 tctacatagt ttgatattga ttaaatctgt tcttgatcac taaaaaaaaa aaaaaaaaaa
     1261 aaaaaaaaaa aaaaaaaaa

• 
  

7/17

Finished moving tank and put it into place. Almost completely finished with plumbing just a few more connections to finish up tomorrow. Tank was filled with tap with bleach to sanitize. will drain and refill with saltwater when all plumbing is in place and leak free. Currently no leaks.




Revision to Methods
Instead of just having a sand bottom to the tanks and Trash cans Jon recommend that I fill 2 gallon buckets that are 9" deep with sand and place them throughout the tank so that when the specimens need to bee transported I can bring up the bucket with sand hopefully not disturbing the specimen to much and transfer to a trash can. The specimen will be in the same substrate that it is used to hopefully making it easier to take samples.
Will also make it easier to collect substrate.

7/16

Primer information for coelenterazine

was amplified by PCR with primers containing the sequence encoding NdeI site, a bacterial periplasm localization signal (pelB) and XhoI site at the 5ʹ end and PstI site at the 3ʹ end.

Anaesthetizing

Cnidaria folks and vwlau

 For sea anemones, I pour an isotonic, fresh-water solution of 7 1/2%
Epsom Salts = magnesium sulfate (from the drugstore - that’s the hydrated
form, with 7 chemically associated water molecules, not the chemical reagent
stuff without the associated water) into the container with the relaxed
anemones to make a final solution that is about 1/2 seawater and 1/2 mgSo4
solution.  
 The tricks I use are to prevent reaction/contraction by the animals
is to cool the solution to the same temperature and aerate it thoroughly,
and to condition the animal to water motion (by putting it near the water
inflow area), first.
 Good luck!

Liz Francis

http://maillists.uci.edu/pipermail/cnidaria/2004-March/000767.html 

http://www.google.com.ni/patents/US6436682

7/15

A meeting with Jake cleared up a lot of the confusion in the procedures of my research.

Found a book with realted information to the system used for biolumenescence in the orange sea pen

Photoproteins in Bioanalysis edited by Sylvia Daunert, Sapna K. Deo looking into picking it up from UW libraries.

Methods- working on expanding need to speak with Dr. Roberts to discuss his intended kits so I can download info on use of each.

Animal collection and care

Tissue Sampling  (Magnesium Sulfate Anesthesia?)

DNA Isolation (Qiagen DNEasy Kit, DNAzol method, etc)

Pick primer for PCR

PCR

RNA Isolation

cDNA

PCR/qPCR

Manipulation experiment  

Luminescence Quantification

RNA/PCR/qPCR

Gene Sequencing?     

7/14

Attended Lab meeting online had a few technical difficulties but I believe I have them all worked out if I can not make the meeting in the future I can Attend online and be able to interact.

Spoke with Jon about setting up the enclosure this week. We are still waiting to hear back from Greg about being able to use the Aquarium room to house the enclosure. Greg replied and said it would be fine so Jon and I will get everything up and running thursday at 1. He plans to move the Tank early tomorrow and we can hopefully have it up and running by thursday.


Started working on the proposal but I will need to speak with Dr. Roberts about some parts of it . Sent an email to schedule a meeting with him as well as Jake. (update-going to put together questions for Dr. Roberts and include them in an e-mail after meeting with Jake)

Created google Doc "https://docs.google.com/document/d/15zQqsOAc0LP0_hbktm-GUAUWwspLUh6cN15MgJzoEng/edit?usp=sharing" to make proposal more accessible

Created possible questions for research

1. What is the genetic sequence and proteins responsible for luminescence in P. gurneyi.

2. Does changes in P. gurneyi environment effect its ability to produce light when touched. (not sure on setup yet, but ocean acidification may be best fit since it has a direct effect on levels of Calcium often used in luminescent reactions but still need to verify this.)

 

07/10

Heard back from Tim Carpenter the curator of fish and invertebrates at the Seattle Aquarium.

• He recommeded 1.25-1.5 gpm for the flow rate and designing a chaotic flow rate instead of direct flow.
• Keeping the Pens between 48 to 53 degrees to slow down algal and any other potential problem growths.
• They feed them Artemia nauplii daily he said anything that would be used to promote coral or planktivorous anemones.
• Dim lighting
• 8" for sand bed is what his are kept in

He also confirmed alki pipeline and reef are good locations to find them. Every once in a while they can be found at golden gardens. 

( North of the Alki Fishing Reef if you have a boat or can scooter from Laurelhurst beach, 40-50 ft depth.

At the end of the Alki Pipeline (at 63^rd Ave. south of Alki point) – long swim out, about 40 feet depth.

Faye Bainbridge Park, Bainbridge Island – 30-40 feet, easy shore access.

Twin Spits near Port Gamble – easy shore dive. )

Asked if he had any knowledge of the resilience to research since I will need to take samples for the genetic, an protein area of my research. 

7/9

Meeting with Jon to start putting together a plan to setup enclosures. Found one ~200 gallon tank that should fit the needs of the project. He recommended that we setup in aquarium room but need to speak with Greg to verify. Tank is current located in storage in salmon hatchery and will need to be transferred over. Jon is not available to do so until next week so we are planning to move everything then. Will need to locate equipment to setup overflow and add circulation to the tank.

Jon also recommended finding a different food supply since culturing my own is not plausible for the size of experiment. I noticed in my reading (posts 7/7) that the Pens may not actually feed on phytoplankton but mainly detritus so their are many options to look into.

Learned I cannot collect specimens by myself because of permit needed. Uw has a permit but only certain individuals are covered under that permit. Will speak with Greg to see if he is willing to collect specimens for me. May need to look into getting a collection permit for myself for current and future research.

7/8

Meeting with Dr. Roberts
Reviewed lab calendar, lab wiki, archiving data, google+, lab meetings, blog posting and signed contract.

Joined Northwest dive club.

Unable to dive with Sonia but got in contact with some of her connections and found optimal dive sites with confirmed sitings of specimens (4 mile barges, fox island west wall, Sunny-side south of pipeline, Les Davis, Whidbey, Bainbridge island as well as alki reef.) Alki reef is supposed to have tons of them but I am not sure about acquiring specimens since Alki is a no take zone. Reviewed and signed lab contract.

requested access to lab wiki, and signed in a created document folder on school hard drive for storing files.

Need to make appointment with Sam White when he returns to go through lab safety and lab tour.

Scheduled Meeting with Jon to go over and plan setup for aquarium, as well as feeding resources for specimens.

Proposal due next Friday, Dr. Roberts will be in Friday Harbor so it needs to put as priority for next week.

Personal notes:
Rottifers may not be a valid food supply, Jon said he had problems with them in the past looking into an alternative may be nessacary refer to 7/7 blog post.

7/7

Species selected (Ptilosarcus gurneyi)

Aquarium Needs: large sand/Mud bed (species full grown average 2 feet, collect specimens 1' or smaller)
Species needs constant water flow (filter feeder, planktonic food supply needed)
Sump system for water circulation possible breeding ground for Plankton (copepods? Tigriopus californicus)

• Sump
• Aquarium
• pump
• overflow box
• intake circulation powerheads/circulation pump
• growth lights for sump and aquarium (add sand to sump as well as local marco algae for copepods)
• mechanical filtration?

Decide to house in Roberts controlled Temp room or possibly talk with Greg about housing it in the Aquarium room so I could connect in to his system which likely already has a large supply of planktonic food available circulating in the system. 

Species collection Locations Bainbridge island, Whidbey island, Alki (spoke with LFS in the area no one knew of any good locataions but Greg has said that Bainbridge was a good location to find plus read (http://www.wallawalla.edu/academics/departments/biology/rosario/inverts/Cnidaria/Class-Anthozoa/Subclass_Alcyonaria/Order_Pennatulacea/Ptilosarcus_gurneyi.html ) an article stating that they are often found "behind whidbey island"

Got in contact with Jon Wittouck <wittouck@uw.edu> to put together

 everything needed for aquarium setup

Update: Jon replied and told me we should not have any 

trouble getting everything set up raised a good question

 to check "biomass of plankton filtered out of water per day" to establish feeding needs

Species creates luminescent mucous?
Species needs large sandbed or lower stalk can becomen inflamed.

T.carpenter@seattleaquarium.org-e-mail sent requesting information on Sea pen needs and possible diving sites.

LFS
BARRIER REEF renton(copepods)
1717 NE 44th St, Renton, WA 98056  (425) 277-7670

Saltwater city, bellevue(copepods)
14150 NE 20th St F3, Bellevue, WA 98007  (425) 644-7050

The fish store, lake city
12320 Lake City Way NE, Seattle, WA 98125  (206) 522-5259

Denny's pretty world, Kirkland
12534 120th Ave NE, Kirkland, WA 98034  (425) 821-3800

Sierra pets, renton(copepods)

601 S Grady Way, Renton, WA 98057  (425) 226-3215

http://www.advancedaquarist.com/2003/10/inverts

Basic Aquarium handling and care 

Diurnal pattern of expansion

Culture of Rotifiers recommended or artifical plankton SDMP (ESV's spray-dried marine phytoplankont), APR (Artificial Plankton - Rotifer), or "Size I" (50-100µm), Cyclop EEze if grouded up to small bits.  Feeding on a regular basis

Check other types of plankton as better food supply

Online research shows mechanical filtration better for cold water systems since the cold temperature slows metabolic rates making it difficult to establish bacteria to properly cycle tank, and local live rock not porous enough to act as a good biological filter. (Check if tropical live rock/dead rock could be used as a replacement).live rock is a good habitat for copepods as well as macro algae (join aquarium blog to ask about cycling coldwater aquariums and types of filtration)

Passive Suspension Feeding in a Sea Pen: Effects of

Ambient Flow on Volume Flow Rate and

Filtering Efficiency

BARBARA A. BEST'

Department of Zoology, Duke University, Durham, North Carolina 27706

Reference:Biol.Bull.175:332-342. (December, 1988)

http://www.biolbull.org/content/175/3/332.full.pdf

Flow rate: 

Small optimum 5-6 cm/s data shows optimum efficiency at 1.5 cm/s

Mediuim/large 8-9 cm/s 

data shows optimum efficiency at 1.5 cm/s for both all sizes change from 1.5 to 6.0 decrease efficiency from 42.8-29.9%

optimum food particle size 10-25 microns

Height stats: sea pen with a rachis height of 7 cm, medium sea pen 15 cm high, and large sea pen 25 cm high

http://www.itis.gov/servlet/SingleRpt/SingleRpt?search_topic=TSN&search_value=52424

Kingdom Animalia  – Animal, animaux, animals  

Subkingdom Radiata   

Phylum Cnidaria Hatschek, 1888 – cnidarians, coelenterates, cnidaires, coelentérés, água viva, anêmona, caravela, cnidario, coral, hidra  

Subphylum Anthozoa   

Class Anthozoa Ehrenberg, 1834 – corals, flower animals, sea anemones, anémones de mer, coraux, água viva, anêmona, antozoário, caravela, corais, gorgônia  

Subclass Octocorallia Haeckel, 1866  

Order Pennatulacea Verrill, 1865 – sea pens, sea panzies, sea pens  

Suborder Subselliflorae Kükenthal, 1915  

Family Pennatulidae Ehrenberg, 1828  

Genus Ptilosarcus Verrill, 1865  

Species Ptilosarcus gurneyi (Gray, 1860)

http://data.gbif.org/species/2258713/?extent=-129%2B41%2B-109%2B51&zoom=5&minMapLong=-129&minMapLat=41&maxMapLong=-109&maxMapLat=51&c[0].s=20&c[0].p=0&c[0].o=2258713

http://www.asnailsodyssey.com/LEARNABOUT/PEN/penFeed.php

Consolidation of research information 

the food particles are unhatched brine-shrimp Artemia cysts