S-STEM Scholar Evan Russell's Blog

  Introduction     We prepared twitch media this week! Chad was kind enough to provide plates for twitch. The pink "barbie" plates should slightly change color as NADH is metabolized upon the media. Additionally I prepared some trusty 0.75% skim milk agar (at 1.0% agar). I placed molds within the media to create inoculation sites and sites in which I can place attractants.  Methods Twitch Motility Assay Protocol 1) Select media (e.g. 0.75% SMA and 1% agar) 2) Prepare a sterile hood by wiping everything down with ethanol 3) Melt media down 4) Wipe down all inoculating materials and insert them into the sterile hood 5) Dispense 10 ml of media into empty petri dishes containing five pronged molds to allow for insertion sites a. Use a sterile 10 ml pipette 6) Let plates cool 7) Remove molds 8) Use UV light for 15 minutes to ensure sterilization of plates  9) Inoculation center of plate (directly onto the plastic) 10) Dispense 100-150 microliter...

Introduction     Welcome back everybody! This week we got the lab up and running again. Our main objective was to get our cells passaged from freezeback. Additionally, we began mixing and autoclaving media so that we would have plates and broths to work with. This year we decided to use slants instead of plates for stock for the added benefit of utilizing slants over the course of three months when plates would only ever keep cells fresh for maybe two weeks.      This year in the world of twitch media, we are experimenting with different dyes within the agar to better visualize the movement of cells. Updates coming next week on dyes that we decide to utilize.  Methods Twitch Motility Assay Protocol 1) Select media (e.g. 0.75% SMA and 1% agar) 2) Prepare a sterile hood by wiping everything down with ethanol 3) Melt media down 4) Wipe down all inoculating materials and insert them into the sterile hood 5) Dispense 10 ml of media into empty petri dis...

  Introduction Last week of lab for the year of 2024. Everything is winding down. Time to make more freeze backs and toss out expired media. We are also going to give presentations on our projects and where we wish to go from here.  I helped out another group with their RNA isolation this week. They are attempting to get an RNA sequence of Deserti bacterial cells that have been desiccated. The process requires meticulous planning and can easily go awry.  Methods Morph Assay Protocol 1) Inoculate mother plate with sample 2) Inoculate R2A and TGY Plates and Broths 3) Incubate Plates and Broths a. Some broths will be shaken and some kept still  4) Gram Stain Plates and Broths after at least 24 hours 5) Record and compare results 6) If needed, introduce a protease to break apart pili connections 7) Re gram stain after 24 hours 8) If needed, run an RNA sequence to check gene expression Twitch Motility Assay Protocol 1) Select media (e.g. 0.75% SMA ...

 Introduction We are going to attempt the Pro K morph assay again. This time we are using 40ug/ml which is four times the amount used in the original protocol. Hopefully the increased concentration will increase our chances of cell dissociation.  Additionally we are going to look at twitch plates that have had attractants added in one quadrant with the hope that it will show directed twitching.  Methods Morph Assay Protocol 1) Inoculate mother plate with sample 2) Inoculate R2A and TGY Plates and Broths 3) Incubate Plates and Broths a. Some broths will be shaken and some kept still  4) Gram Stain Plates and Broths after at least 24 hours 5) Record and compare results 6) If needed, introduce a protease to break apart pili connections 7) Re gram stain after 24 hours 8) If needed, run an RNA sequence to check gene expression Twitch Motility Assay Protocol 1) Select media (e.g. 0.75% SMA and 0.5% agar) 2) Prepare a sterile hood by wiping everyth...

  Introduction The use of proteinase K has been inconclusive thus far and so we have decided to increase the concentration to 20ug/ml. Proteinase K is optimized at 50-65 degrees Celsius so its activity is slowed at the incubation temperature of P81. Hopefully increasing the concentration will account for the decrease in temperature.  Additionally, lab is closing soon for winter break and winding down protocols are in effect. Media production has been halted to as needed only and freeze backs of samples are being created.  Methods Morph Assay Protocol 1) Inoculate mother plate with sample 2) Inoculate R2A and TGY Plates and Broths 3) Incubate Plates and Broths a. Some broths will be shaken and some kept still  4) Gram Stain Plates and Broths after at least 24 hours 5) Record and compare results 6) If needed, introduce a protease to break apart pili connections 7) Re gram stain after 24 hours 8) If needed, run an RNA sequence to check gene express...

  Introduction The plans for twitch and morph were put into effect this week. We studied the effects of proteases upon the connected P81 cells while simultaneously studying the effects of chemoattracts upon twitching P81 cells.  Methods Morph Assay Protocol 1) Inoculate mother plate with sample 2) Inoculate R2A and TGY Plates and Broths 3) Incubate Plates and Broths a. Some broths will be shaken and some kept still  4) Gram Stain Plates and Broths after at least 24 hours 5) Record and compare results 6) If needed, introduce a protease to break apart pili connections 7) Re gram stain after 24 hours 8) If needed, run an RNA sequence to check gene expression Twitch Motility Assay Protocol 1) Select media (e.g. 0.75% SMA and 0.5% agar) 2) Prepare a sterile hood by wiping everything down with ethanol 3) Melt media down 4) Wipe down all inoculating materials and insert them into the sterile hood 5) Dispense 10 ml of media into empty petri dishes co...

  Introduction This week we looked into proteases for the morphology assay. Proteinase K, Trypsin, Pepsin and carvacrol are often used to break down biofilms through different methods. Proteinase K at 10 ug/ml has been used for other species and so we will apply it here. The idea is that if the connection between cells is facilitated purely by pili connections, the dissolution of strings to rods after the application of Proteinase K will provide support for this theory. However, if the connections between cells are the result of incomplete cell division, then no separation should take place after the introduction of Pro K.  For the twitch runs, we are introducing the chemoattractants: fumaric and Malic acid. Chemoattractants bind to twitch capable cells and ignite a pathway that results in cells twitching in the direction of the chemical gradient of attractants. Thus, if the cells are indeed twitching, they will be compelled to twitch in the direction of the attractant to ...

  Introduction The twitch and morph assays were the assays performed this week.  The twitch assay looked at different forms of media to see if there were any differences in twitch based upon media type (in this case, pure tryptone vs pure yeast vs pure dextrose). For the morph run, we compared TGY and R2B again to try to get consistent results with last week. We are trying to confirm that media type (more so than environmental condition) is responsible for the morphological change in which P81 rods begin to connect end to end. Furthermore, a morph run in which we begin to apply Proteinase K took place this week. This enzyme can break down the pili between cells that forms biofilms. This week we began to calculate proportions for our stock solution.  Methods Morph Assay Protocol 1) Inoculate mother plate with sample 2) Inoculate R2A and TGY Plates and Broths 3) Incubate Plates and Broths a. Some broths will be shaken and some kept still  4) Gram Stain ...

  Introduction For the morph assay, gram stains are used to identify whether the P81 rods are taking the form of single rods or connected rods. Different conditions such as shaking or stirred or TGY vs R2B are used to determine if nutrient density or environmental stability effects the ability of these rods to connect. As an aside, with the amount of gram stains necessary for the morph assay, it is wiser to place several samples (up to four I’ve found) upon a single slide to save time and materials.  For the twitch assay, we discussed the possibility of using different dyes to be able to better visualize the twitching cells. Dyes discussed include ONBG and XGAL. Methods Morph Assay Protocol 1) Inoculate mother plate with sample 2) Inoculate R2A and TGY Plates and Broths 3) Incubate Plates and Broths a. Some broths will be shaken and some kept still  4) Gram Stain Plates and Broths after at least 24 hours 5) Record and compare results 6) If needed, introd...

  Introduction Within the morph project, it was observed that P81 cells tend to age more in TGY than in R2B. This could be a result of the increased nutrient content causing enhanced metabolic activity and increased replication speed. Furthermore, the gram staining process for the morph project has been streamlined and the observations are more thoroughly and effectively documented.  The twitch runs were further documented and the pictures have been uploaded to a google doc.  Methods For the morph project, the cells were packed to get better gram stains. Morph Assay Protocol 1) Inoculate mother plate with sample 2) Inoculate R2A and TGY Plates and Broths 3) Incubate Plates and Broths a. Some broths will be shaken and some kept still  4) Gram Stain Plates and Broths after at least 24 hours 5) Record and compare results 6) If needed, introduce a protease to break apart pili connections 7) Re gram stain after 24 hours 8) If needed, run an RNA seq...

 Introduction We did another run of the morph project. It was difficult to obtain high quality gram stains last week so we are adjusting our approach and increasing staining efficiency.  The twitch projects are ongoing. It takes approximately 10-14 days to get results with this assay. Meanwhile Nick and Riyah are working on their UV projects and Andrew is working on his Salt survival project.  Methods Morph project – From both an R2A plate and a TGY plate: two TGY plates, two R2A plates, two R2B broths and two TGY broths were inoculated. 24 hours later these sources were gram stained. Twitch project – Sugar water attractants. 1 gram of dextrose per 1000ml. This solution was poured into an indentation within the media plate to be used as a chemoattractant. Morph Assay Protocol 1) Inoculate mother plate with sample 2) Inoculate R2A and TGY Plates and Broths 3) Incubate Plates and Broths a. Some broths will be shaken and some kept still  4) Gram Stai...

  Introduction The twitch project looks at the twitching motion of Deinococcus aquaticus P81 cells as they spread along a plate of media in response to metabolites in the environment. Using minimal media may help prompt cells to twitch to a greater extent as they spread in search of nutrients.  The morphology project looks at the elongation of P81 when they are grown in broths instead of upon a plate. The current theory is that the cells are connecting end to end via pilli connections. We are looking into environmental and media conditions that may induce a morphological change. We are currently comparing shaking vs still conditions as well as TGY vs R2B broth.  Methods Morph project - From a stock TGY plate we inoculated a R2A plate, TGY plate, two R2B broths and two TGY broths. One of each of the broths was placed in a shaking incubator while the other was placed in a still incubator. Twitch project – we inoculated skim milk plates of 0.5% skim milk and 0.75% sk...

  Introduction Following a lack of growth on the experimental plates of the latest transformation run, we are currently shelving the transformation of Deinococcus aquaticus P81. We are now focusing on the twitch assay and the morph assay. The twitch assay focuses upon the movement of Deinococcus aquaticus P81 via twitching mechanisms. Bacteria with pili extensions can drag themselves across surfaces, thus allowing them to move in search of nutrients even when they lack a flagella. This assay involves placing the bacteria directly onto plastic and tracking their movements over several weeks as they move towards the surrounding media in search of food.  Additionally we are performing a morph assay. In this experiment, we are tracking a morphological change that we observe in P81. P81 elongates when grown in broth. The current hypothesis for this phenotypic change is that P81 forms a sort of biofilm in which the pili connections at the polar ends of the rods are connecting to...

  Introduction Another transformation attempt was undertaken for P81. Varying amounts of plasmid were utilized again to see if altering the concentration might increase the chances of transformation. Methods 5 microliters of 220 ng plasmid was used for the first experimental group. 7 microliters of 220 ng for the second and 10 microliters for the third. Transformation Protocol 1) 1 ml cells OD value of 1 (late log) 2) Pellet 3) Resuspend in 100 microliters of TGY 4) Add 40 microliters of 0.3 CaCl2 mix 5) Ice 15 minutes 6) 30 microliter sample and about 10 microliters of plasmid (where the plasmid is 200-400 ng per microliter for a grand total of around 1 microgram of plasmid DNA) 7) Mix 8) Incubate at 30 degrees Celsius for 90 minutes while inverting every 15 minutes. 9) Add  2 ml of TGY and Incubate for 24 hours 10) Plate cells on TGYA and TGYA CM plates 11) Use Cells mixed with PCR water as a blank Results Growth on the control plates and no growt...

  Introduction The transformation procedure was attempted again this week with a particular interest upon the OD value. We are attempting to perfect the procedure in every conceivable way so that any failure for P81 to transform can not be attributed to human error. Additionally, varying amounts of plasmid were used across experimental groups to test whether plasmid concentration has any effect. Methods A 278 ng per microliter plasmid extraction was used. The first experimental group was given 4 microliters while the second experimental group was given 8 microliters. The final experimental group was given 12 microliters of a 220.4 ng per microliter plasmid extraction.  Transformation Protocol 1) 1 ml cells OD value of 1 (late log) 2) Pellet 3) Resuspend in 100 microliters of TGY 4) Add 40 microliters of 0.3 CaCl2 mix 5) Ice 15 minutes 6) 30 microliter sample and about 10 microliters of plasmid (where the plasmid is 200-400 ng per microliter for a grand total of...

  Introduction This week, another attempt was made to transform Deinococcus aquaticus P81.  Methods We packed the cells 3x this week to see if an increase in cell concentration would help the transformation process.  We also prepared some new 0.3M calcium chloride. Transformation Protocol 1) 1 ml cells OD value of 1 (late log) 2) Pellet 3) Resuspend in 100 microliters of TGY 4) Add 40 microliters of 0.3 CaCl2 mix 5) Ice 15 minutes 6) 30 microliter sample and about 10 microliters of plasmid (where the plasmid is 200-400 ng per microliter for a grand total of around 1 microgram of plasmid DNA) 7) Mix 8) Incubate at 30 degrees Celsius for 90 minutes while inverting every 15 minutes. 9) Add  2 ml of TGY and Incubate for 24 hours 10) Plate cells on TGYA and TGYA CM plates 11) Use Cells mixed with PCR water as a blank Results Growth upon the control plates and a lack of growth upon any chloramphenicol plate.  Discussion Many avenues wer...

  Introduction This week we were performing gram stains for the morphology project. This project looks at two different conditions: type of media and whether that media is inoculated while shaking or remaining still. Deinococcus aquaticus P81 cells have been observed to “elongate” when inoculated into a broth instead of upon a plate. The current theories explaining this morphological changes is that P81 might be attaching end to end by a biofilm or that P81 under mobile conditions fails to completely separate.  This week we are inoculating multiple broths of TGY and R2B and observing whether or not the cells will continue to elongate as expected.  Simultaneously, we are running another transformation procedure with P81 to attempt to insert the PRAD1 plasmid into P81. A successful transformation would give transformed P81 cells resistance to both ampicillin and chloramphenicol.  At the same time we will be inoculating another run of SMA plates for the twitch m...

  Introduction Plasmid extractions are performed upon E. coli cells containing the PRAD1 plasmid that contains the genes for both chloramphenicol and ampicillin resistance. E. coli is easy to grow and is thus a useful vector for the PRAD1 plasmid. The plasmid can be replicated and stored by maintaining E. coli colonies on LB plates containing ampicillin. The ampicillin is necessary within the LB plates to maintain an environmental pressure for the E. coli to hold onto the PRAD1 gene. Without this environmental pressure, E. coli would eject the PRAD1 gene as bacterial cells are extremely energy efficient and do not hold onto genes that are not necessary for survival.  A zyppy kit DNA retrieval protocol (and kit) can be used to lyse cells and extract the plasmid DNA within. The extracted plasmid can be used in a transformation procedure. The plasmid can potentially be inserted into a new species which would then allow for the genetic expression of the plasmid DNA within the ...

  Introduction Transformation of Deinococcus aquaticus strain P81. The transformation of Deinococcus aquaticus has been attempted by many students in Dr. Tuohy’s Biotechnology Lab without success thus far. The P81 strain, containing certain pili genes that the reference genome lacks, was hypothesized to perhaps have a better chance to transform. Transformation, or the uptake of external DNA into a cell, is partially facilitated by pili genes thus giving us hope that this strain will transform more easily than the reference genome.  Methods A TGY flask of P81 is selected. Samples of P81 are aliquoted into 1ml portions contained within Eppendorf tubes. One Eppendorf tube is set aside for gram staining and for finding an OD value. After obtaining a clean gram stain and procuring an OD value near 1 (In this case the OD value was 1.32). 1 ml samples of P81 were aliquoted into three more Eppendorf tubes.  We pellet these cells. We resuspend the cells in 100 microliters of T...