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Vt Marushchak


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Vibrio research progress report

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Vt Marushchak

  1. 1. Vibrio tubiashii research update Tatyana Marushchak
  2. 2. Foot Experimet <ul><li>1 colony of bacteria was added to the 10mL of the marine broth. 4 tubes were made: clam foot tissues was added into 2 tubes. All the tubes were placed in the incubator for 72 hours. The temperature was set to 17C and speed to 175. After 48 hours, foot tissue was removed out. </li></ul>Protein Gel: distinct bands in the samples without foot; smear in the samples with foot, but differnt size bands evident- possible proteases Gel was repeated – same results Isolation of the RNA: There is significantly more RNA in the foot samples mass of the pellet: VT+foot-------0.86g VT no foot---0.22g Spectrophotomer analysis: VT+foot-------1669.37 ng/ul Vt no foot-----351.01 ng/ul Both samples were diluted to the concenration of app. 150 ng/ul PCR of the samples: Primers used: 1. Vtubi_16s 2. Vtubi_chitinase 3. Vtubi_vthB VthB and Chitinase produced bands. Interesing absence of 16s bands
  3. 3. Gigas Hemocyte Challenge <ul><li>I have not done anything with these samples. </li></ul><ul><li>I. 4 ml of marine broth (MB) were split into 4 different tubes. 2 of the tubes received 1 ml of hemocytes, while other 2 tubes, 1 ml of sea H2O. 1 tube with the hemocytes and 1 tube with sea H2 O were inoculated with 1 colony of VT. Same steps were repeated using 4 ml of Sea H2O instead of MB. All tubes were incubated for 24h, 18C @100RPM. Samples were collected by spinning at 100g, 18C, 5 min </li></ul><ul><li>II. 5 ml of MB was mixed with either 0.5 ml of hemocytes or sea H2O. Each sample was prepared in duplicates, one for control and one for experiment. 1 colony of VT bacteria was added to the experiment tubes. Each sample was plated on Poly-D-lysine plate, incubated at 18C, 100 RPM for 18h. Supernatant was collected and spun, pellet resuspended in 1ml of Tri reagent. Plates were rinsed with 1 ml of Tri reagent. </li></ul>
  4. 4. Exposure Experiment <ul><li>Four tanks were set up at RT. Three tanks received 4.435 X 10^11 Vibrio tubiashii bacteria (32ml bacteria/tank). Each tank was randomely allocated among 3 different treatment: tank 1- sterile water, tank 2 -alive oysters and sea water, tank 3 - autoclaved oysters and sterile water. 4th tank was set up as a control and contained alive oysters in sea water WITHOUT Vibrio tubiashii bacteria. Two 1 ml samples were taken from each tank for RNA/Protein analysis. Samples were drawn at: 0, 2 hours, 4 hours, 21 hours and 24h </li></ul><ul><li>Protein Gel: samples at t=4 and t =21h--- as expected sample with autoclaved oysters had more Vt thus more protein. Based on the photos though it did not seem much higher than Vt grown in sterile seawate </li></ul><ul><li>Protein Quantification by Coomassies Assay: Spectr.analysis C autoclaved > C alive > C sea H2O </li></ul><ul><li>2D Protein Gel: </li></ul><ul><li>Isolation of the RNA: concentration is higher in the presence of hemocytes </li></ul><ul><li>qPCR: t=24 samples---Chitinase and vthB generated a signal on qPCR (however no big differences.) FliM had no products. Vtubi_16s generated a signal–no big difference. </li></ul><ul><li>PCR: primers used ToxR, ompW, fitsz--- No bands </li></ul>
  5. 5. PH experiment <ul><li>This experiment is designed to check responses of V. tubiashii to different pH values. 1 ml of V. tubiashii bacteria (C=1.386x10^10 bac/ml) was added to 19 mL of either marine broth or sea H2O of different pH. Marine Broth pH Sea H2O pH 7. 62 8.57 6.62 7.57 8.61 9.56 All tubes were put on the shaker and temperature was set to 20C. 1 mL samples (2) were taken after 5 min, 30min, 1 hour, 2 hours and 24 hours. </li></ul><ul><li>Protein gel: Sea H2O samples, t=30 sec-----No dramatic difference in effect of pH </li></ul><ul><li>RNA isolation: Sea H2O, t=2 h–Concentration at pH 7.57 is twice lower than at pH 8.57 </li></ul><ul><li>qPCR: Sea H2O samples, t=30 sec----Chitinase and vthB look produced signal (however no big differences). FliM had no products. t=2---Vtubi_16s generated a signal </li></ul>
  6. 6. Specificity of primers <ul><li>Primers: Vtubi_16sV2, Vrpos, tdh,Vspa24, Vphil_contig854,Vtubi_chitinase, Vtubi_VthB, VP21,22 were checked for spcificity </li></ul><ul><li>Primers were tested on : Vibrio parahemoliticus, Vibrio vulnificus and Vibrio tubiashii </li></ul><ul><li>Results: Vtubi_VthB is species specific Vtubi_chitinase does not replicate </li></ul><ul><li>NOTE: some of the primes did not produce a band when one was expected. This could have happend because cDNA was too much diluted) </li></ul>
  7. 7. Seed Experiment -1 st slide <ul><li>Two poly-D lysine plates were seeded with 3.25mL Gigas hemos. Incubated 12C ~16hrs in the dark. 1mL Vt culture grown o/n at RT in MB(~28C, @ ~175RPM). After 12 hours, plates were gently washed with sterile sea water. 3 mL sea water added to plates after washing. 1 plate innoculated with Vt (200uL), one with MB (200uL), and a third plate just had Vt added (200uL in 3mL sea water).Incubated 3 hrs. at 12C in dark @ 50 RPM. Plates with Vt = supe. collected, pelleted, remove supe, resuspend pellet in 1mL Tri-Reagent. Plates with hemos = supe. removed, washed plates with 1mL Tri-Reagent. </li></ul><ul><li>qPCR: primers used: QPX-SPB, SR4-SER-PRO, VRPos, Ser-Pro, Vtubi_16s, R-TDH, VP22 +VP21 </li></ul><ul><li>RNA isolation: C hemos+Vt> C vt > supHemos+vt > only hemos. </li></ul><ul><li>PCR: primers used: 18sgigas, Vtubi16 SF All samples including H2O(control) samples turned out with the band of the same size </li></ul><ul><li>Results of qPCR: SPB primers – nothing SR4 SER PRO -- possibly only with hemocytes (plated and solution) maybe increase anneal temp SerProB -- possibly only with hemocytes (plated and solution) Vtubi16s -- high signal with Vt. low signal in hemo withoug Vt. -increase temp R-tdh -- signal only with non-adhered hemocytes VPP22,21 (tdh) -- 1st four. just hemo, just Vt, with hemo VRPOS (VP16s) -- nothing </li></ul>
  8. 8. Seed Experiment-2 nd slide <ul><li>PCR of selected samples that were done on the qPCR: nothing relevant–we should redo it with differnt anealing T. We used anealing T of 50C, while anealing T at the qPCR was 55C. </li></ul>
  9. 9. Temperature Experiment <ul><li>4 tubes of marine broth were prepared. Two tubes were treated with 1 colony of Vibrio tubiashii bacteria. 2 other tubes served as control. All tubes ( treated with VT and control) were plated in the incubator with temperature of 12C or 21C. </li></ul><ul><li>Isolation of the RNA: Spectrophotometric analysis showed that Vt grown at 21C contains more RNA. C12C = 0.235 microg/microL C21C = 1.37 microg/microL </li></ul><ul><li>PCR: primers used: Vtubi_chitinase Vtubi_VTHB OmpA OmpB Vtubi_chitinase and Vtubi_VTHB produced a band as expected. OmpA primer pair proced a band. </li></ul>