Team:ColumbiaU NYC/Notebook
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May
5/22-5/28
We solidified the synthetic mosquito repellent idea. We spent the week conducting a literature review to determine what research has been conducted on the rhamnolipids and bacteria strains. We pinpointed a few subtopics that we could pursue such as freeze drying and skin grafting. We divided the research among ourselves and put together a powerpoint containing a summary of our ideas to present to our faculty advisors. After the meeting, we made a comprehensive project timeline schedule and made experimental plans for the following week.
5/29-6/4
Molecular Cloning
We started molecular cloning with a PCR amplification of the RhlAB operon from the P. aeruginosa P14 genome extract. For the screening assay, we used a superfolded GFP. With our faculty members, we discussed whether we should clone using EcoRI restriction enzyme cuts or Gibson assembly. Initially, our PCR was unsuccessful because the primers we designed had too few base pairs annealing to the sequence. After ordering the correct primers, we used Gibson assembly to insert the gene into the backbone, which was successful. Additionally, we transformed plasmids without the RhlAB insert into P. putida KT2440. Then, we picked colonies with different GFP intensities to obtain a library of different constitutive promoters as well.
Qualitative Analysis of Rhamnolipids
Regarding the production of rhamnolipids, we researched a few different tests before deciding to try the CTAB qualitative test with pure rhamnolipids. Furthermore, we decided to try freeze-dry P. putida to determine what percent of the bacteria would be viable after extended periods of time. We could use this knowledge to determine shelf-life of the mosquito repellent for commercial applications.
Mosquito and Mouse Experiments
We reached out to several researchers as well in preparation for future experiments. We contacted Dr. Voshall at Rockefeller University who does mosquito testing. We want to determine what concentration of mono-rhamnolipids and di-rhamnolipids actively repel mosquitoes. We also reached out to Dr. Christiano who works with mice to discuss whether we can use her laboratory to conduct rhamnolipid experiments using human skin grafted on mice. In preparation for the meeting, we drafted a set of experimental protocols and created a Powerpoint presentation.
Community Outreach
We planned a community outreach project in which we can administer a survey to individuals to determine if they are highly attracted or unattracted to mosquitoes and then use next generation sequencing to sequence samples of the skin biome to deduce if there are any commonalities.
June
6/5-6/11
Freeze Dry
The initial freeze dry experiments were successful but we realized that we need a more quantitative way to analyze the success of the freeze dry. Perhaps, we need to incorporate a survival rate or ratio calculation. We decided that we need to also extend the timeline for the freeze dry experiments to more closely mimic real world situations.
Rhamnolipid Quantification
We conducted the CTAB experiments with the rhamnolipids and blue halos were present. However, we realized CTAB only works well for higher concentrations than what we need to produce. Furthermore, we would need a more quantitative metric than qualitative one. We brainstormed and figured that we could use an image application to measure the diameter of the halos and correlate that to the concentration of rhamnolipids.
Community Outreach
We had several meetings this week one of which was with Dr. Shaman, a skin swabbing expert, to discuss our idea for community outreach. Dr. Shaman had collaborated with the Museum of Natural History to swab skin biomes in the skin biome exhibit. He suggested that we avoid collaborating with the museum due to the extensive IRB process and provisions of the board of trustees. He also cautioned our timeline and monetary expenses.
Keratinocyte Experiments
We met with Dr. Prince who is a skin bacteria specialist to discuss collaboration. We planned to conduct experiments to determine how the bacteria strains and rhamnolipids affect skin cell viability. Dr. Prince liked our idea and put us in contact with a graduate student in her laboratory to gather more information. She agreed to provide her lab space, human skin cells (keratinocyte), cell media, and other reagents to aid in our experiments.
6/12-6/18
Molecular Cloning
Our Gibson assembly worked for two different promoters; we now have two constructs transformed in E. coli including a strong promoter from Streptococcus pneumoniae and a weak promoter from Propionibacterium acnes. We started to work on the construction of BioBrick parts. We began to build BioBricks for different promoters for the RhlAB operon. We made plans to characterize the promoters in E. coli, P. aeruginosa, and P. putida. This week, we also continued experiments to transform recombinant plasmids into P. putida using electroporation. We contacted Dr. Jiang from the Marraffini Rockefeller University to obtain the S. epidermidis strains, recommended plasmids, and shuttle vector for transformation/conjugation. He also sent us specific protocols to utilize with this strain.
Rhamnolipid Quantification
We conducted more CTAB experiments in which we made test plates with different concentrations of rhamnolipids to make a standard curve using the diameters of the halos for our quantitative measure. Unfortunately, we determined that the halo diameter varies with the thickness of the CTAB on plates in that thicker plates give bigger halos for the same concentration of rhamnolipids. This characteristic created inconsistency across different CTAB plates. There were no visible halos for low concentrations of rhamnolipids indicating that we might need to calibrate CTAB to detect lower concentrations of rhamnolipids. We also conducted the CTAB test using live E. coli cells with the RhlAB recombinant plasmid, but there were no visible Halos possibly due to low Rhamnolipid concentrations. The overall conclusion is that the CTAB test may be used as a qualitative analysis, but is not reliable as a quantitative method due to condition-dependent halo diameters. We found a different assay called an orcinol assay that may be used in place of the CTAB test.
Freeze Dry
We focused on counting the exact levels of survival using cell count methods.
Mosquito Experiments
Because the lab at Rockefeller University did not respond to emails, we contacted the Fidock lab at Columbia University. The Fidock lab has extensive experience with Malaria experiments and has incubators that they would be willing to allow us to use. We made a meeting for the following week and began to draft experimental plans to discuss with Professor Fidock in person.
6/19-6/25
Molecular Cloning
We received S. Epidermidis strain RP62A, shuttle strain S. Aureus RN4220, a plasmid pC194 from S.A. RN4220, a plasmid pC221 and pGO1 from S.A. OS2 from Dr. Jiang at Rockefeller. We will start cloning next week once we design the primers.
Freeze Dry
Experiments were conducted using M9 minimal media or LB as the rehydration media. The lyoprotectants used was either 10% Sucrose or 10% Sucrose with LB. Based upon the analysis conducted in the freeze dry research paper, survival rate was calculated. The highest survival rate calculated was 2.25% using LB as the rehydration and the 10% Sucrose lyoprotectants.
Rhamnolipid Quantification
As a way of detecting rhamnolipids, we decided to try thin-layer chromatography (TLC). For different staining methods, we tried orcinol with 50% sulfuric acid, orcinol with 10% sulfuric acid, CAM, and KMnO4. We discovered that orcinol with 10% sulfuric acid gives the best visibility, and the limit of detection was determined to be 0.5 mg/mL. We were also able to distinguish between mono-rhamnolipids and di-rhamnolipids through TLC analysis.
Mosquito Experiments
We met with Professor Fidock who agreed to let us work in his laboratory. He put us in contact with a mosquito seller from NYU and a trained epidemiologist with whom we can discuss the experimental setup. However, the epidemiologist will not be available until August 1st. Thus, in the meantime, we will communicate via email regarding the experimental protocol and setup.
6/26-7/2
Molecular Cloning
We made glycerol stocks of Dr. Jiang's strains and mini prepped plasmids using lysostaphin. We began the process of building recombinant plasmids using Gibson assembly. In addition, we successfully created a BioBrick with a weak promoter that we transformed into P. putida. We are still working on removing illegal cut sites in RhlAB operon. We also began the documentation and characterization process of our parts.
Transformation
At this point, we have a weak promoter construct working, yet we are still having difficulty transforming a strong promoter in order to produce a greater degree of rhamnolipids. We believe that this may be the result of a metabolic burden. We brainstormed a few possible solutions to this problem such as using conjugation methods or an inducible promoter.
Rhamnolipid Quantification
This week we tried to increase the sensitivity of the CTAB assay by using less CTAB. We discovered that the detection limit was still too high. It was difficult to tell if the halos were due to rhamnolipids or GFP already present in the cell culture. Regular LB also showed halos as well, reinforcing evidence that this assay should not be used.
TLC was utilized to quantify rhamnolipids first using a small batch and then a larger batch of P. putida. Using the smaller quantity of P. putida, there was too little rhamnolipid to detect. For the larger batch, cell culture was grown for a longer period of time and purified via liquid-liquid extraction. Still the quantity of rhamnolipid was too small to detect. We decided that we should use supercritical fluid chromatography mass spectroscopy (SFC-MS) to increase sensitivity for measuring rhamnolipid quantity.
Freeze Dry
We repeated the freeze dry experiments using different storage times: immediate and 3 days. In addition to calculating survival rate, we also calculated the bacterial survival ratio. The paper we were using as a model reported an 80% "survival rate." In reality, the survival rate they reported was a different metric called bacterial survival ratio. This difference in terminology explained why we thought our experiment was not working. We had a 2.26% survival rate which corresponds to an 82.34 bacterial survival ratio, which is the number the paper reported. Thus, our results are on par with the paper.
PhaC Knockout
We ran a PCR on the PhaC gene in knockout P. putida from Spain against PCRed PhaC gene of WT P. putida to confirm if a transposon was indeed in the PhaC gene. Lengths of the PhaC gene in both strains were the same, indicating the transposon was not in the PhaC gene.However, a PCR product created from transposon-internal primer sequence provided in the paper existed for the knockout but not WT, indicating that the transposon was somewhere in the knockout.We will do a full sequence of 200 bp upstream of PhaC gene to 200bp downstream PhaC gene in the knockout to get clearer picture of what is going on.
Mouse and Mosquito Experiments
We began the process of IRB approval and spoke with Dr. Christiano who suggested we speak with Dr. Owens about establishing the IRB. We also registered for lab safety training using mice. We met with a lab member from Dr. Owen's lab, Rong Du, to discuss the IRB in more detail and gave her a copy of our experimental protocols. We also improved our protocol upon discuss with a graduate student, Lekha Nair, from Dr. Christiano;s lab.
July
7/3-7/9
Keratinocyte Experiments
We began our first keratinocyte experiments in Dr. Prince’s laboratory. The goal of the experiment was to determine which MOIs of the bacteria strains (Putida, Mutant, Arginosa) were toxic to the cells and to establish whether the bacteria was engulfed by the cells by plating. The experiment took longer than expected as we included two incubation points: 2 hours and 4 hours. We then had to leave the laboratory as there was no longer a qualified scientist available to watch us. Before leaving, we placed the cells on slides in preparation to count them in a different laboratory for later use. However, by the time we got to the other laboratory the cells had died. We learned from this experiment that we should not make the slides too far in advance. We also learned that we should use a legitimate cell viability assay rather than a crude counting method. Finally, we realized that we should split the large experiment into smaller experiments to determine dosage and time point this way we will have enough time to accurately complete both experiments. Then we can use the optimal conditions determined by these experiments in a future culminatory experiment.
Rhamnolipid Production Troubleshooting
Seeing as it seems that there were no Rhamnolipids being produced by our strain from the MS and TLC data we decided it would be best to break down the process to a step by step level and trouble shoot at each level. To test our hypothesis that there was a metabolic burden, we agree it would be best to see if a high concentration of rhamnolipids is toxic to cell growth and test an inducible promoter. We also thought that there may be necessary reagents to grow such as glucose. So, we decided to replicate the exact conditions of the primary paper from which we were working. We questioned the promoters as well. Should we clone the natural promoter of RhlAB into P. Putida. Furthermore, is the bacterial strain an issue? To answer the lattermost question, we decided to check if transformed E.Coli produces Rhamnolipids along with P. Aeruginosa. To analyze the transcription step we ran a qPCR and to assess the translation step we ran an SDS-Page.
Based upon the TLC analysis, we made the following conclusions. First, we Confirmed that purification of rhamnolipids via liquid-liquid extraction and TLC analysis work efficiently for cell culture as well. Next, we Confirmed that PAO1 mostly produces di-rhamnolipids. Lastly, both E. Coli and P. putida do not produce enough rhamnolipids for TLC analysis. Comparing the MS peaks from P. Arginosa, pure rhamnolipids, and E.Coli + RhlAB, the peak induced by our construct was not sufficiently pronounced to conclude that rhamnolipids were produced.
S. epidermidis Experiments
After adding 5-fold inserts instead of the usual 3, we successfully PCRed out all the parts necessary to form the two recombinant plasmids needed to perform Gibson Assembly. Filter conjugation of empty pC221 into S. epidermidis RP62A was successful. We made glycerol stocks of electrocompetent S. Aureus RN4220, OS2/pGO1 and S. Epidermidis RP62A cells. We made plans to electroplate empty vector and construct in the coming week. To ensure that S. epidermidis could survive the secretion of rhamnolipids, we conducted a qualitative survival test by directly plating S. epidermidis with the necessary 1g/L rhamnolipids for Mosquito repellent activity. The resultant colonies appeared more sparse in rhamnolipid-Epidermidis cultures than cultures with Just the bacteria. In order to determine the effect of rhamnolipids on already established colonies, we grew S. Epidermidis on plates overnight, and then added 1g/L rhamnolipids to the already present colonies. A day later, the colonies in the after one day rhamnolipid addition plate were slightly denser than those in the immediate addition of rhamnolipid plate, but less dense than the plates that had absolutely no rhamnolipids present.
Freeze Dry
We analyzed the difference between survival rate and survival ratio to get a better sense of the difference between the two metrics. We conducted the experiment again with the four day incubation. We planned future experiments in which the incubation time would be extended to about a month.
Mouse and Mosquito Experiments
Given a sample IRB from Dr. Owens we drafted our own IRB using our protocols and sent it to Dr. Owens for review.
Community Outreach
Because the next generation sequencing is highly expensive, we decided for our community outreach that this may not be the best idea. Additionally, we would have to undergo training for human subjects experiments and establish a new IRB protocol. We still thought it was beneficial to develop a survey and FAQ about mosquito borne illnesses. We drafted a version of the survey asking questions about frequency of mosquito bites, knowledge of mosquito borne illnesses, and preferred characteristics of mosquito repellent (ex: is scent important when choosing a repellent?). We sent the survey to a cohort of individuals as a pilot study to find any errors in the survey data and gain preliminary results. The power analysis revealed that we need about 250 participants or more to obtain statistically significant results.
7/10-7/16
Rhamnolipid Quantification
We grew wild type P. Aeruginosa and transformed P. Aeruginosa with L1 + RhlAB, and extracted rhamnolipids from the two strains using liquid-liquid extraction. SFC-MS result shows that mutant P.A. produces much more mono-rhamnolipids, confirming that our construct works well in P. Aeruginosa. We also tried growing mutant Putida and E. Coli (transformed with L1 + RhlAB and H2 + RhlAB) in a different growth media, LB + glucose. SFC-MS result shows that all of them make mono-rhamnolipids: E. Coli + H2_RhlAB made the most mono-rhamnolipids, then E. Coli + L1_RhlA, then putida + L1_RhlAB.
Transformation of P. putida PhaC knockout
We tried transforming the knockout strain with 4 different promoters and RhlAB, but only L1+RhlAB showed GFP positive colonies. We will sequence it to confirm the operon and quantify the amount of rhamnolipids.
Protein Analysis
We ran the gel, but it was unclear and blurred because we did not purify the gel. However, there were bands of the right size for RhlA and RhlB. MS showed that rhamnolipids were detected. So, we decided not to spend the time purifying the gel.
qPCR
Results show that mRNA is being properly synthesized by our construct, although our negative controls (wild-type and empty vector) indicated some unexpected mRNA.
Keratinocyte Experiments
We obtained our cells, but they were highly confluent during the weekend. We performed experiment testing effect of different bacterial strains (paK, WT PP, PP+L1+Rham, and WT PP with 1g/L mixed rhamnolipid) with different MOIs (10, 100, 1,000, 10,000, 100,000) on keratinocyte viability. Cell survival was testing with the MTS cell viability assay, which measures the level of live cells per sample. Keratinocytes were first plated at 3x10^4 per well into 48 well plates, then weaned off P/S antibiotics on the next day. The following day, the different strains were each added with the different MOIs with 3 trials per situation. Positive control was a "0 MOI" situation with no bacteria added, which should represent maximum cell viability, and two negative controls were pure media and pure media with 100,000 MOI of each bacterial strain, which should give a cell viability of 0. This was repeated at 2 time points: 3 and 24 hours. Data from the 3 hour time point has been collected.
Molecular Cloning
We tested the Staphylococcus protocols for miniprepping, electroporation, and filter-mating with empty vectors. We also constructed our recombinant pC221+RhlAB+GFP and pC194+RhlAB+GFP plasmids for transformation into the Staph strains. To more closely observe the effect of Rhamnolipids on S. epidermidis, we performed a gradient plate experiment which once again showed sparser colonies for S. epidermidis with higher concentrations of Rhamnolipids. We decided that designing a quantitative experiment with a 96-well plate, instead of just relying on qualitative experimentation was necessary.
7/17-7/23
Metabolics Troubleshooting
Given the low yield of rhamnolipids from the successfully cloned promoters of our promoter library, alternative promoters were explored to possibly boost production. Experiments were planned to more closely replicate the Wittgen paper by extracting the RhlAB operon with its natural promoter by PCRing out the operon along with 200 extra bps upstream. Cloning this new operon had limited success and rather than repeating experiments it was deemed less relevant. More priority has been placed on designing/extracting the Xylose inducible promoter given that it might be viable for usage in Staph epidermidis as well.
Promoter Characterization
Most of the higher strength promoters have been unreliable in terms of transformation and rhamnolipid production. It is speculated that the issue with this lies in that they are constitutively triggering the RhlA gene to make the rhamnolipid precursor HAA while the RhlB is only active during the stationary phase. Buildup of HAA might be causing the bacteria to view the operon as impeding survivability and in some way shut it down. This could be solved using the Xylose inducible promoters so that the buildup of HAA is prevented and high levels of rhamnolipids would be produced upon induction in the stationary phase.
Keratinocyte Experiments
Keratinocytes were treated with different MOIs of bacteria strains Putida, Arginosa, Mutant, and Putida with Rhamnolipid to determine the relationship between bacterial concentration and cell viability. The cells were treated with gentamicin after 3 or 24 hours and then an MTS assay was utilized to quantify cell viability. The cells were then plated to deduce if bacteria had been drawn in by the cells. The results showed that there was not a significant difference in the bacterial concentrations, which may indicate that the serial dilutions were incorrect. Additionally, there was a high degree of bacterial growth on the plates, which was not expected. Perhaps, the gentamicin did not kill the bacteria on the cells either because the concentration was too low or the the incubation time period was not long enough.
Another experiment was conducted to determine the IC50 of rhamnolipid by treating the keratinocytes with different concentrations of rhamnolipids for 24 hours. The concentrations selected were based upon the results of several research papers that utilized rhamnolipids with different cell lines. Graphpad software was utilized to model the the data and it was determined that the IC50 is between 40 and 60 ug/ml.
7/24-7/30
Molecular Cloning
Tired of waiting for the xylose inducible promoter backbone plasmid, we decided to utilize what we have already available in the lab and clone arabinose inducible promoter into our construct. We designed and ordered the primers for the new construct with the arabinose promoter.
Rhamnolipid Quantification
This week, we wanted to see if saturating LB with glucose helps producing rhamnolipids. We grew mutant putida with LB + 10g/L of glucose, 30 g/L of glucose, and 50 g/L of glucose and measured the amount of rhamnolipids at different time points. After 24 hours of growth, 50 g/L glucose gave the most amount of rhamnolipids. After 72 hours of growth, all three growth media gave slightly less rhamnolipids. It is possible that bacteria are eating up rhamnolipids as they run out of carbon sources during the stationary phase. We also created a more accurate standard curve using 95% pure mono-rhamnolipids.
Keratinocyte Experiments
Based upon our suspicion that the gentamicin was not killing the bacteria, we conducted an experiment in which we manipulated the concentration of gentamicin and the incubation time points using four different strains of bacteria (E.coli, W.T. Putida, Aeruginosa, and Mutant strain). The highest time point and concentration was four hours at 200 ug/mL yet these conditions were not successful on all of the strains.
We planned repeat the bacteria MOI experiment with different, accurately calculated MOIs knowing the new information about gentamicin incubation time and concentration. Because the 4 hour time point was unsuccessful, we were unsure for how long to incubate the cells in gentamicin before completing the MTS assay. Thus, we decided to leave the cells in gentamicin overnight and concomitantly treat a separate bacterial culture with gentamicin overnight to compare the bacteria growth.
We encountered another problem with the experiment because our control cells were contaminated by bacteria. We could not bring the bacteria into the cell culture hood so we brought the cells to the bench when we added the bacteria under flame. Even with these precautions there was contamination. After discussion, we decided to continue the experiment by treating with gentamicin overnight, thinking that the gentamicin will kill the contamination in the negative control conditions as well. While this experiment may not yield all the results we wanted (specifically bacteria intake by the cells), we will be able to determine if there is a difference in cell viability given different MOIs.
Transformation of S. epidermidis
We successfully transformed pC194_RhlAB into the shuttle vector for electroporation, S. Aureus RN4220, however transformation of pC221_RhlAB into the conjugation vector, OS2/pGO1, was unsuccessful. We re-tried electroporating pC221_RhlAB into OS2/pGO1 a couple more times, but we were unsuccessful. We also conducted the 96-well S. EPidermidis Experiment, however, the Rhamnolipid blank began to grow around the 7;12 hr mark, making it necessary for us to disregard the results.
7/31-8/6
Metabolics Troubleshooting
We have successfully cloned the arabinose inducible RhlAB backbone and transformed into 10-beta E. Coli strain. We have also transformed it into Putida, which gave us bacterial lawns. Surprisingly, the lawns were GFP positive, and we learned that Arabinose isn't really inducible in Pseudomonas. We will still compare its expression level to our original construct.
Keratinocyte Experiments
We conducted more experiments to determine at what concentration of gentamicin and time point successfully kills the bacterial strains. However, they were unsuccessful again. We spoke to our mentors and decided that a biofilm may be forming that prevents the bacteria from dying. We conducted OD experiments to determine at what point the bacterial strains are in mid exponential phase. The results were inconclusive for all of the bacterial strains except for E.Coli. Thus, we tried to conduct an experiment to assess which MOIs of E.Coli cause cell death but there was an error with our cell culture and the cells were dead in the morning. We now think that the problem with our experiments is that we are using very high bacteria concentrations. We made plans to repeat the experiments with lower concentrations.
Molecular Cloning
We successfully electroporated pC221_RhlAB into S. Aureus RN4220. We intend to electroporate mini-prepped pGO1 helper plasmid into RN4220, and then filter conjugate from here, since we were unable to electroporate pC221_RhlAB directly into OS2 which already contained pGO1. After Millipore filtering our Rhamnolipids solution, we repeated the 96-well S. Epidermidis vs. Rhamnolipids survival test. Our negative control was S. epidermidis with 2uL of 34 ng/uL Kanamycin, while our positive control was S. epidermidis with just BHI. After adding a 1/1000th dilution of overnight to a range of two-fold serial dilutions from 2000mg/L to 15.6mg/L. The resultant OD600s showed that growth of S. Epidermidis is staggered by higher concentrations of Rhamnolipids up to125mg/L of Rhamnolipids, although the OD600 Does eventually get to OD 1 by the 12th hour. So although production of Rhamnolipids in the high concentrations we need might stagger the growth of S. epidermidis, it will not necessarily kill our cells.
IRB Update
We have submitted a draft of IRB amendments to IACUC, which we are adding on to Dr. Owens protocol. Since we are changing Dr. Owens’ pain level to D, as a result of the skin-grafting component of our mouse experiments, we are getting a vet approval which will be sent to the IACUC as well this week. We have also been assigned an IACUC consultant, Sierra Fuller, who has been advising us on the level of detail that must be included in the protocol.
Marketing
We have started thinking about how we can market our mosquito repellent. Using AutoCad, we will 3D print our marketing bottle!
August
8/7-8/13
Metabolics Troubleshooting
It looks like there was a recombining when we transformed the arabinose inducible promoter plasmid into Putida. Even though the colonies were GFP positive, sequencing result gave us a negative result.
Keratinocyte Experiments
This week the cells died because they were contaminated with bacteria. We plated the cells to confirm our hypothesis. We contacted the laboratory that gave us the cells. The graduate student will not have them available for another few weeks because their cells were also contaminated. Thus, these experiments will be put on hold. However, we decided to try a different assay technique in which we lyse the cells and then measure bacteria presence. This will avoid the complications regarding the concentration and duration of gent application to the cells.
Community Outreach
We created a map based upon the survey data illustrating what regions of the country experience more mosquito bites than others. We collaborated with the University of Georgia and assisted them on their project. We also contacted BioBus an organization in which scientists teach students about the field while they are on a bus to plan an event with them. However, the scientists will not be back to work until September. We made arrangements to contact them in September. Additionally, we made contact with the Chinese iGEM Team who is putting together a newsletter and asked if we would like to contribute.
8/14-8/20
Mosquito Experiments
Last Friday, we sourced Aedes Aegypti mosquitoes from the Vosshall lab at Rockefeller University, who kindly gave us two large tupperwares full of larvae and pupae and braved NYC traffic to drive us back to CUMC. The mosquito life cycle consists of 4 stages: eggs, larvae, pupae, and adult. On Saturday, Sunday, Monday, and Tuesday, we sorted out the male mosquitos (cannot suck blood) in both pupal and adult stage. The pupae are sexed by size (females are larger) and adults are sexed by antenna (males have fuzzy antennae while females do not)[insert picture] We spend countless hours in the cold room sorting adult and pupal mosquitoes because the cold prevents mosquitoes from escaping.
Keratinocyte Experiments
This week we conducted an experiment using smaller bacterial MOIs to determine if the high MOI was the reason that gentamicin was ineffectual. All four bacterial strains were plated at MOIs that do not exceed 10. The following day there was no evidence of bacterial growth! Thus, when the new keratinocytes are received this concentration of gentamicin can be utilized in the experimental procedure.
Community Outreach
We arranged an event scheduled for October 1st with the organization ThinkSTEAM that promotes science and math education in girls. The program will consist of a presentation introducing systems biology, research overview, and laboratory demonstrations.
8/21-8/27
Keratinocyte Experiments
This week we obtained the flask from the Prince Lab because they resolved the microplasma problem. I fed the flasks and split them. However, when I came back to the lab after the weekend there were fungi growing in the flasks. I checked the incubators and there is a warning that filters need to be replaced, which may explain the contamination. I notified the laboratory of the contamination issue. I contacted other laboratories in the building to see if we can use incubators while waiting for the incubators to be replaced and one laboratory agreed.
8/28-9/3
I was able to plate cells this week and conduct an experiment to determine if E.Coli causes cell death. We utilized a new essay from a paper entitled, "Internalization of Pseudomonas aeruginosa Strain PAO1 into Epithelial Cells Is Promoted by Interaction of a T6SS Effector with the Microtubule Network." We utilized the gent concentration specified in the article and lysed the cells prior to plating. The experiments revealed that the bacteria did not cause significant cell death at any concentration. Additionally, when the cells were plated bacterial colonies did not grow, indicating that the bacteria was not internalized by the cells.
