1. Progress Report 8/26/14

2. Projects Color Choice Behavior Assay Expression of iGluRs in Xenopus Oocyte

3. Linear Relationship between Intensity and Digital Input

5. Using Python to create GUI (graphical user interface) Use GUI to easily set conditions in light box

6. Green Phototaxis 0.00253 0.0253

7. CS Color Choice

9. Variables to Consider Circadian Rhythms Allada & Chung (2010) Circadian organization of behavior and physiology in Drosophila

10. Variables to Consider Age (5-7 days) Temp Humidity (80% best) Amount of time provided for color choice decision

11. Positive control lines UAS RNAi for Clumsy II UAS RNAi for Clumsy III Tm5c-G4 -> TNTE Dm8-G4 ->TNTE Negative Control lines UAS RNAi for Kainate R1C III

12. Cross Parental- UAS-Dcr2; ortC1a-G4DB#3#4 OK371dVP16AD; + Cyo + X UAS RNAi (chrom. 2 or 3) UAS RNAi

13. F1F1 UAS-Dcr2; ortC1a-G4DB#3#4 OK371dVP16AD; + UAS RNAi + OR UAS-Dcr2; ortC1a-G4DB#3#4 OK371dVP16AD; + + UAS RNAi Choose: Non-Cyo Non- Tm3 Sb Non- Tm6 Hu

14. Tm5c-G4 -> TNTE Parental: +; UAS-TNTE x yw; ortC1a-G4; ortC1a-G4 Cyo TM2 F 1 : +; UAS-TNTE; ortC1a-G4 yw ortC1a-G4 + Y

15. Dm8-G4 -> TNTE Parental: +; UAS-TNTE x yw; ortC2b-G4; ortC2b-G4 Cyo TM2 F 1 : +; UAS-TNTE; ortC2b-G4 yw ortC2b-G4 + Y

16. Candidates for screen Goal: Identify Novel Kainate Receptor Auxiliary Proteins Auxiliary Proteins are are characterized by the following criteria: 1. Do not serve as an integral component of the transduction pathway 2. Remains stably associated with the receptor it regulates 3. Affects multiple aspects of receptor pharmacology, function and subcellular trafficking or targeting 4. Co-assembly with receptor is necessary for proper receptor functioning Copits&Swanson(2012)

18. Expression of iGluRs in Xenopus Oocyte Infusion HD Cloning Kit – Cloning the following iGluR into pT7Ts vector: CG5621, DKaiRIC CG9935 CG3822_DkaiRID CG11155 Clumsy Inject mRNA from assembled clones into Xenopus Oocyte

19. Primer Design Ting designed primers to amplify iGluRs – Fw_infusion_1: TTTGGCAGATCTGATATC XXXXXXXXXXXX – Re_infusion_2: TCAGTCACTAGTGATATC XXXXXXXXXXXX Primers contain 15 bp region complementary to ends of linearized pT7Ts

20. CG5621,DKaiRIC: – Template: pOT2_CG5621_GH10694_DkaiRIC (2469+36=2505) – xCG5621_infu_F: TTTGGCAGATCTGATATCATGTGGTTCGTGAAAATGATTTCAACA – xCG5621_infu_R: TCAGTCACTAGTGATATCCTAATCTCCAGGGCCACGG CG9935 – Template: pUAST- CG9935 (2700+36=2736) – xCG9935_infu_F: TTTGGCAGATCTGATATCATGTTAATAGCAAGCGGCTTCTTAC – xCG9935_infu_R: TCAGTCACTAGTGATATCTCATAGTTTATTAATTCGTTGGCCTTTCG CG3822_DkaiRID – Template: pFLC-I-RE_CG3822_DkaiRID (2562+36=2598) – xCG3822_infu_F: TTTGGCAGATCTGATATCATGCGGTCGAGTGGAGTTT – xCG3822_infu_R: TCAGTCACTAGTGATATCTCATTCGTTCAGTATCGCGCT – CG11155 – Template: pFLC-I-FI_01405_CG11155 (2733+36=2769) – xCG11155_infu_F: TTTGGCAGATCTGATATCATGGTACGAAAAAAACGAGAAATCGT – xCG11155_infu_R: TCAGTCACTAGTGATATCTTAATTAAAGTAATTATAGTAAACGCCCACATTTGA Clumsy – Template: pCRB-clumsy_F1-2_R3-2_no9_good (3009+36=3045) – xClumsy_infu_F: TTTGGCAGATCTGATATCATGTATTCTCTATTCCTCACTCACTTTCTT – xClumsy_infu_R: TCAGTCACTAGTGATATCTTACTCAGGAGGCGAGGGA

21. Prepare pT7Ts vector: 1.Culture E.coli at 37 C, shaking, overnight in LB/Amp 2.Harvest pT7Ts using Promega plasmid mini-prep 3.Linearize pT7Ts through EcoRV digestion 4.Gel Purify Prepare iGluR inserts: 1.Amplify template with primers containing 15bp homologous extensions 2.Gel Purify

22. 1 kb ladder pT7Ts CG5621 CG9935 CG3822 Clumsy 2.5 kb 3 kb 2 kb 3kb 1kb 2 kb 2.6 kb 1kb

23. 1 kb ladder CG5621-Colony1 CG5621 - Colony2 CG5621-Colony3 CG5621-Colony4 3 kb 1kb 2 kb CG9935-Colony1 CG9935 - Colony2 CG9935-Colony3 CG9935-Colony4 CG3822-Colony1 CG3822 - Colony2 CG3822-Colony3 CG3822-Colony4 3 kb 1kb 2 kb CLUMSY-Colony1 CLUMSY - Colony2 CLUMSY-Colony3 CLUMSY-Colony4 Primers: M13F and M13R

24. Construct M13R T7 5’ UTR 3’ UTR Sp6 CG5621; 2505 bp CG9935; 2736 bp CG3822; 2598 bp CG11155; 2769 bp Clumsy; 3045 bp M13F 5’ 3’ SmaI

25. To Do: Cloning of CG11155 into pT7Ts Sequencing of positive clones