Measurement

Overview

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project 1: quantifying how blood effects reporter strength

research question: how does blood effect reporter strength? how well can we detect reporter (i.e. chromoproteins or fluorescent proteins) signals within diluted blood?

run 2: voetir dark fake blood, 1-1, 1-3, 1-7, 1-9, water (in order fromof left to right) (left number is blood, right number is water)

initial fluorescence experiments: a potential end product for tnbc patients utilizes a fluorescence measurement of mirna’s 21 and 155 circulating in blood to provide a quantitative result via the minipcr “biobits” kit. we wanted to test what the lowest dilution level with blood added that fluorescence is clearly visible. for samples one through three, the addition of blood renders the fluorescence undetectable or close to it. the small quantity of what appears like fluorescence is caused by a reflection of the light coming from below. proof will be provided later on. there is slight fluorescence detectable in the 1-3 sample, too slight to be distinguishable from basal levels in blood. based on these results, we must use a minimum a 1-7 dilution. importantly, this means we need to lower the detectable threshold of our measurement target (mirnas 21 and 155) to a value lower than our target in standard whole blood.

run 1: from left to right: 1:9, 1:7, 1:3, 1:1, 0:1

distilled water on the left, water sample from run 1 on right.

explanation of glare: when we initially ran the test for fluorescence penetration in blood, we had a concern that the water sample fluoresced at a lower intensity than our two most dilute blood samples. in theory, water fluoresces at the highest rate. as such, we hypothesized that the inducer for the biobits was simply never added to the water sample in run 1. to test our hypothesis we simply prepared an identical biobits tube, and purposefully left out the iptg inducer. as pictured above, the two samples were in fact identical. therefore, we created a new run (run 2) and ensured the inducer was added. thankfully, we had the expected results for our water sample in this run. you will notice that even in the samples without the inducer, we still see fluorescence, however this is actually glare caused by the light shining from below the tube.

depiction of blue-based chromoprotein samples before + after addition of 20 ul blood sample

depiction of base rgb values of chromoproteins from plates

blue toned chromoproteins blood addition writeup: the goal of this experiment was to see if we could see a color change when there is a combination of chromoprotein and blood. we wanted to avoid a green chromoprotein, which would produce a brown mixture with blood, as a brown color in blood can be caused by other health factors naturally. we chose a blue chromoprotein to create a purple mixture when mixed with blood. this unique, unnatural color would then be used to distinguish between positive or negative target mirna quantity test results. we added a pink chromoprotein as a form of negative control, as a pink and red mixture are similar enough colors that they wouldn’t result in a detectable change (assuming we have performed the procedure correctly). however, we were unable to produce distinguishable results after adding the blood sample. we suspect this is due to adding too much blood to a too-dilute chromoprotein sample. additionally, cells have a tendency to settle back into pellets out of solution after roughly a minute or two. we have made several attempts to create colorful chromoprotein-producing liquid culture of engineered bacteria, however we have only been able to see color on plates. this makes it hard to standardize the amount of bacteria added, and generate consistent color patterns.

nanodrop od measurement for blue chromoproteins

the readings pictured above were taken at the beginning of our experimental process.

potential for future measurement: due to the challenges we’ve had in our lab trying to develop meaningful results with too low quantities of chromoprotein-rich cultures, we’ve decided to put this experimental series on the sidelines. however, we believe this testing method and process has extensive potential for future igem teams, or future iterations of this project to take advantage of. linked below is a document containing an in-depth experimental plan/protocol for the development of a od based chromoprotein standard, and an ideal experimental pathway to generate meaningful positive results. https://docs.google.com/document/d/1d4kzdrjftvufigwzswkpbrbjizxgfznlqdutxuso8ic/edit?usp=drivesdk

sfgfp as a chromoprotein: considering our difficulties with aeblue, we wanted to use a different chromoprotein, and we landed on sfgfp. we originally avoided gfp, thinking it would be green when exposed to natural light (meaning when mixed with red, it would turn brown). however, when spun down and rehydrated according to the same guidelines as aeblue, we found it to be yellow. this yellow color could produce an orange instead, which matches our criteria of an unnatural color. our primary research question here is: what is the amount of standardized dilute blood it takes to result in a noticeable color change? to answer this question, we first made simple a dilution curve of od to dilution of sfgfp samples, so that our experiment can be repeated. as outlined in the protocol below, we identified our “most dilute sample that is indistinguishably yellow” (or mdsiy), which was our ¼ dilution. then, to actually test for the color change, we set yeast producing b-carotene as the “orange” target. what we mean by target is the rgb values of something known to be orange, whose color our cells would then try to match as we added diluted blood. we chose this as our target, as b-carotene is responsible for orange color in carotene, which is universally considered orange. we wanted to use these yeast as our target instead of a carrot itself for both uniformity of color, and so that any possible influence our test tubes or lighting may have on rgb values are the same between target and sample. we chose to add diluted blood (1 part blood to 7 parts water) to our samples 1 ul at a time. we chose this specific dilution because, as defined in our initial fluorescence experiments, this dilution is the most concentrated with the least amount of dampening effect on fluorescence. this is important, so that we can match fluorescence of our sample’s color in another experiment. we found that 1ul of 1-7 fake blood alone was enough to cause a color change to a pink-red color whose rgb values far exceeded the targets. we moved forward with the remaining stock sample, and our ½ dilution. we found 2 ul were enough to cause a match in the ½ dilution, and 3 ul were enough to cause a change in our stock sample. [need to perform again with more replicates]

protocol: https://docs.google.com/document/d/1d4kzdrjftvufigwzswkpbrbjizxgfznlqdutxuso8ic/edit?usp=drivesdk

project 2: determining if mirna 21 can cross the cell membrane

research question: how much mirna makes it into bacteria cells that express toehold switches? how does that relate to reporter output from the toehold switches?

ct values of ivt mirna with splicer probes: the goal of this experiment is to generate a ct value curve with mirna’s 21, 155, 159, and the probes for each, to be used as a standard for our cell membrane recovery experiments, detailed later on.
exp 1: we used a set of 3 dilutions for each mirna, specifically 1 nm, 1pm, and 1 fm concentrations, reflecting a range of values found in the splicer paper [cite] and literature on concentrations of mirna in circulating blood [cite]. when performing qpcr we generated the following graphs linked below.

https://docs.google.com/document/d/1mskvyfjeerfodp5qwye6hzjfi0hthmms42lgfyhlyek/edit?usp=drivesdk

after analyzing the data, we saw that ct values were very high for all concentrations and with a slight trend towards lower ct values the higher the concentration as expected.

exp 2: given the high ct values from exp 1, we decided to try higher concentrations to see if we can get any signal differences between the concentrations. we also found a paper that uses 10 nm concentrations of mirna when adding to e. coli cultures [cite]. we are now re-running the experiment using concentrations of 1 um, 100 nm, 10 nm, and 1 nm mirna and hope to create a more effective ct curve.

protocol: https://docs.google.com/document/d/1rxxuyuph1yw8lqt5avycvfkf2lt4mj7ydxwzblx0wv4/edit?usp=drivesdk

ct values were lower for some samples than for exp 1. we also once again noticed a slight trend towards lower ct values as the concentration of mirna added increased.

the rest (very work in progress!!!!!! not meant to be used in wiki until finished)

depiction of serial dilutions before vs after adding 20ul samples of aeblue cells spun from tsb culture then resuspended in water.

fluorescence curve in fake blood: the purpose of this experiment is to benchmark cell-based fluorescence against biobits in fake blood. [im just writing, will compress thoughts to be wiki worthy later: we will use the blood serial dilutions, and replace the water in the biobits kit with them. then we will get the fluorescence values at those different levels. what’s important here is that this allows us to measure how much the blood itself is actually affecting the fluorescence. if our cells can’t reach equivalent fluorescence in the same dilution, or exceed it for whatever reason, that will influence our test design. the complexity of cells introduces so many more variables, so if the overall purpose of our blood experiments as a whole is to see how blood affects the fluorescence, we need the biobits as a reference to see how much of a change is because of fake blood itself, and not because of some other biological issue. if someone were to repeat our experiment exactly, and didn’t have the biobits standard, if they encountered an issue they have no “next step” to figure out what’s causing the difference. if they repeat the biobits experiment, firstly they know for sure whether it’s organic (a difference in our cells), or inorganic (a difference in environment, product, etc).]

can mirna-21 cross the cell membrane?: during our literature review, while we were able to confirm mirna-155 was able to cross the cell membrane of e.coli, there is no equivalent for mirna-21. if the mirna was unable to cross the membrane, we would be unable to use a possible cell-based test. the purpose of the following experiment is to see whether mirna-21 can cross the cell membrane. the method we used to test this was by adding a known concentration of mirna into a culture of e.coli, isolating the mirna using a technique called splicer, and then use qpcr to quantify the amount of mirna recovered. splicer is a technique which uses partially complementary nucleic acid probes, which bind to different parts of the mirna. after these probes attach, they are joined together by splintr ligase. at the base of one of the probes is biotin, which is a substance that binds to streptavidin coated magnetic beads. these beads can be pulled out of solution, which is how we isolate the mirna from the cell contents.

rate of cell membrane crossing: the purpose of this experiment is to determine how fast the mirna molecules cross a cell membrane. this is important for our math modeling team to figure out how much time our test would need to fluoresce.

If you've done excellent work in measurement, you should consider nominating your team for this special prize. Synthetic Biology needs great measurement approaches for characterizing parts, and efficient new methods for characterizing many parts at once. If you've done something exciting in the area of Measurement, describe it here!