Secretion Team: Dilution Error
Design: Run a secretion assay where we would determine the optimal time for induction of culture samples with IPTG. Goal was to induce cultures as the bacteria were exiting the log phase of exponential growth that corresponds to an OD600 Reading of about 0.5-0.6. We want to induce during this phase of bacterial growth to maximize protein yield, which in this case is the EGF ligand bound to mEGFP.
Build: Made overnight cultures of each construct for the signal peptide secretion Designed a Secretion Assay Protocol where to outline how we would OD600 measurements of the culture samples at set intervals(4h, 6h, 8h) to accurately determine when the samples were in the process of exiting the log phase of growth.
Test: Took samples of signal peptide cultures and diluted them 1:4 with LB media in cuvette tubes to take OD600 readings. Recorded values in a Google Sheets Document, and when all of the samples were in the OD600 range that defined the exiting of log phase, they were induced with IPTG. Once Induced with IPTG, the cultures were left overnight, and culture samples were taken at 4h, 16h, and 24h after induction. Those samples were prepared with LDS Page to prepare protein gels for Western Blot Analysis.
Learn: For the first 3 Secretion Assays, the OD600 Readings recorded were not multiplied by 5 to account for the 1:4 dilution of the overnight culture samples in the cuvettes. This meant the cultures were actually being induced when the cultures were well into the death phase of bacterial cell growth. This meant that bacterial cells were in the process of lysing, which is why we saw decreased expression of the mEGFP from these samples in the Western Blot. For the rest of the secretion assays, we accounted for the 1:4 dilution of the cultures in LB Media and multiplied those values by 5.
Secretion Team: Improper Antibody Incubation for the Western Blot
Design: Run a Western Blot on selected samples from successful secretion assay with proper IPTG induction. Need to prepare a primary antibody in a 1:1,000 dilution that binds to GFP, as well as a 1:10,000 diluted secondary antibody that selects for the host animal of the primary antibody. Imaging of the Western Blot will show bands that select for the mEGFP in the supernatant of prepared secretion assay samples.
Build: Took 4h, 16h, and 24h time point samples of induced bacteria cultures from successful secretion assay. Prepared dilutions of primary and secondary antibodies using 1X TBS Blocking buffer. Scanned blotting paper from LDS Page Protein Gel Transfer.
Test: Performed Western Blot, where primary antibody was incubated for either one hour at room temperature or overnight at 4 degrees Celsius, then the blotting paper was washed with 1X TBS Buffer 3 times at room temperature before incubated with secondary antibody for one hour at room temperature or overnight at 4 degrees celsius.
Western Blot from 07/22/2023
(Induced at “Wrong OD” of 0.4)
Issues: Blotting at 4 C instead of Room Temperature
Short Antibody Binding Times for both primary and secondary antibody
Samples induced at Wrong OD
Western Blots from 08/01/2023
Induced OD of 0.2 (Gel 2)
Issue: Secondary Antibody only incubated for an hour
Induced OD of 0.7 (Gel 3)
Issue: Blocking (Not Shaking at Room Temp), Primary Antibody Incubation Only an hour
Western Blots from 08/03/2023
Induced OD of 0.7
Did not turn shaker on while blocking at room temperature.
Learn: With the initial blocking step in TBS, we were performing it at 4 degrees celsius instead of room temperature, so the rate of reaction was too slow. The primary antibody was being incubated for one hour in the cold room for the first 5 Western Blots, which meant that the antibodies did not have enough time to completely probe the blotting paper since the rate of reaction was too slow, which resulted in very faint/no protein bands due to there not being enough antibody bound to protein. Secondary antibody is diluted so much more than the primary antibody that incubating it overnight with the blotting paper overcame the issue with the antibody not binding to the target protein by giving the antibody more time to bind to the protein of interest.
Adhesion Team: Optimizing Adhesion Pair
Design: To discover the most optimal nanobody antigen pair that displays the strongest adhesion, we utlized plasmids created by Dr. Glass. The plasmids borrowed included a C-tag nanobody and antigen and a GFP nanobody and antigen. The plasmids containing the C-tag component had an arabinose (Ara) inducible promotor, which is needed to induce protein expression. Likewise, the GFP plasmids possessed an anhydrous tetracycline (ATc) induicible promoter.
Build: To incorporate the borrowed plasmids into our chosen E. coli, we transformed the desired plasmids into DH10Bs.
Test: Once we incorporated our plasmids into DH10B E. coli cells, the bacteria were then grown for a certain period of time and induced with either ATc or Ara. Bacterial cultures were then mixed together and tested for aggregation through measurements of the mixed cultures supernatant. This test determines whether the antigen nanoboy pairs are successful in adhering to each other.
Learn: Our results showcased strong adhesion between the C-tag antigen and nanobody cells as seen by the large clumps of aggregated bacteria and decreasing measurements of the optical density of the corresponding supernatant. The GFP antigen and nanobody cells, however, displayed no adhesion. Additionally, images of the GFP nanobody and antigen mixtures displayed signs of very low fluorescence and no adhesion as there is little presence of clumps of fluorescent bacteria in Figure 1. Since the GFP antigen was fluorescing at small intensity, we hypothesized that the GFP antigen was faulty. An experiment that confirmed our intuitions was mixing GFP nanobody cells with purified GFP. Our subsequent image showcased that when the GFP nanobody adheres to free GFP, the nanobody fluoresces. Hence, one can conclude that the GFP antigen initially used was problematic.
Inter-Kingdom Team: Tuning Base Parameters for Microscopy
Design: A suitable co-culture environment for both bacteria and mammalian cells.
Build: Plate the mammalian cells on a 96-well plate and incubate for at least 2 hours. Dilute bacterial cultures at various concentrations into PBS and pipette over mammalian cells.
Test: Image co-culture experiments on microscope.
Learn: Media type: We first tried diluting the bacteria into PBS, but it caused the mammalian cells to shrink up and skewed microscopy observations. We did not attempt to co-culture the mammalian cells with bacteria in LB media, as we predicted that it may contain proteins that could activate the ERK pathway separately from our secreted EGF and skew our results. Concluded with using DMEM/FBS mammalian culture media. This required us to make specialized media with different antibiotics in accordance with the bacterial antibiotic resistances, rather than only using stock media.
(Left) HEK293T initial “ruffling” response to PBS-diluted bacteria, (right) response after 25 minutes.
Oil on top of wells: We attempted an overnight time series in which the mammalian cells died after about 4 hours due to media evaporation (gif here). As a result, we started pipetting mineral oil over the overnight co-cultures to prevent evaporation.
Multiplicity of infection (MOI): MOI, or the approximate number of bacteria per cell, was a parameter we had to tune in order to ensure that both the bacteria and mammalian cell could thrive in co-culture. We set up trials at MOI 10, 100, 300, and 500 and ultimately determined that MOI 100 is optimal for overnight microscopy.
Inter-Kingdom Team: Addressing Fluorescence Problems (Visualizing Bacteria)
Design: A way to distinctly visualize both bacteria and mammalian cells together on a microscope.
Build: Transform E. coli with a plasmid that causes a fluorescent (in this case, with GFP) protein to be expressed.
Test: Image bacteria on confocal microscope to assess their visibility.
(Left) GFP-displaying bacteria in green fluorescence channel, (right) same bacteria in transmitted light channel.
Learn: Using a fluorescent marker was crucial to visualizing the bacteria more clearly (compare fluorescent image (left) to grayscale (right)).
Inter-Kingdom Team: Addressing Fluorescence Problems (Separating Bacterial Fluorescence from Mammalian Cell Fluorescence)
Design: In order to distinguish bacteria from mammalian cells (which fluoresce in red and green), we chose to label our engineered bacteria with blue fluorescence.
Build: Co-transform our functional bacteria with a new fluorescent labeling plasmid (mCerulean).
Test: Perform co-culture imaging.
HEK293T cells with membrane red and green, co-cultured with mCerulean-labeled bacteria.
Learn: The mCerulean-labeled bacteria appeared in the green channel (see clumps on cell membranes) of the microscope because our blue laser is not tuned to the exact wavelength that stimulates fluorescence from mCerulean. Thus, the mCerulean labeling effort was unsuccessful given equipment constraints.
Inter-Kingdom Team: Finding bacteria-mammalian cell adhesion pairs
Design: Make lists of all available constructs in human cells (HEK293T line) and E. coli, then create as many nanobody-antigen pairs as possible
Bacteria/mammalian cell pairings.
Sample well plate diagram.
Build:Plate appropriate HEK293T cell lines in a 96-well plate, then allow to grow to about 50% confluency. Pipette engineered bacteria on top of human cells at various concentrations.
Test:Run overnight microscopy, imaging wells every 10-15 minutes.
Learn: Endocytosis hypothesis: Bacteria localize near cells with high GFP expression (brightest membranes). Additionally, some bacterial clumps appear orange rather than green–we formulated a working hypothesis that these clumps had been engulfed by the cells and appeared orange because they were covered by the red-fluorescing cell membrane.
Anti-GFP expressing HEK293Ts + GFP-displaying
(Top left) HEK293T with surface-expressed GFP (membrane red/green) + surface-displaying anti-GFP (green). (Top right) layered Z-image at another point in the same well. (Bottom) Control: blank bacteria.
GFP lysate binding: While we observed some bacterial localization, it was not as dramatic as with the other successful adhesion pair. We hypothesized that there were issues with bacteria displaying GFP, since it is such a large protein. Furthermore, while the bacteria themselves did not adhere to the HEK293Ts, we still observed outlines of the human cells in the green channel (see figure). We hypothesize that GFP from lysed bacteria is binding (competitively) to the anti-GFP nanobodies on the HEK293Ts.
(Left) Anti-GFP HEK293Ts + GFP-expressing bacteria. (Right) Green channel only.
Buggy adhesion pairs:
HEK293T anti-GFP + E. coli GFP: One of our adhesion pairs (anti-GFP-displaying human cells + GFP-displaying bacteria), which we were expecting to show strong adhesion, did not work. We hypothesized that the version of GFP initially in the bacterial construct, avGFP, is not properly synthesized by E. coli, and swapped it out with a new GFP, eGFP.
HEK293Ts + EGFR nanobodies: EGFR (epidermal growth factor receptor) is a receptor naturally found on human cells. However, our EGFR-nanobody displaying bacteria (verified via aggregation assay) did not bind to the human cells. We hypothesize that our HEK293T strain does not express enough EGFR on its surface and that there were not enough places for the E. coli to bind. In future experiments, we would use HEK293Ts which overexpress EGFR to test these E. coli constructs.
Inter-Kingdom Team: Debugging Bacteria-Mammalian Cell Adhesion Pairs: Mammalian Anti-GFP + Bacterial GFP
Design:One of our adhesion pairs (anti-GFP-displaying human cells + GFP-displaying bacteria), which we were expecting to show strong adhesion, did not work. We hypothesized that the version of GFP initially in the bacterial construct, avGFP, is not properly synthesized by E. coli, and swapped it out with a new GFP, eGFP.
Build:Replace the avGFP-displaying bacteria with a different type of GFP (eGFP). Design a construct for a fusion eGFP with intimin autotransporter protein (based on adhesion team’s work) and transform it into E. coli.
Test:Repeat the adhesion co-culture experiment with anti-GFP-displaying HEK293T cells and the new GFP E. coli.
Learn:After switching out the GFP, we observed some adhesion, though it was inconsistent (see next cycle). Furthermore, an aggregation assay with this version of GFP (see adhesion team) was unsuccessful, and the secretion subteam also encountered similar issues when attempting to secrete GFP. Lastly, we also confirmed that the anti-GFP HEK293T cells were working properly by incubating them with pure GFP. This indicated that the bacteria have a problem displaying properly folded GFP on their surface (potentially due to the size of GFP). Future iterations of the engineering cycle would allow us to optimize protein linkers and look more closely at the intimin autotransporter system.
(Left) anti-GFP HEK293Ts incubated with 1uM purified GFP, (right) control - blank HEK293Ts with GFP.
Anti-GFP expressing HEK293Ts + GFP-displaying(?) bacteria.
References
Buch, T. and Rollová, M. (2019). Bacterial Growth Curve by OD600 and SoloVPE. Biofactory Competence Center p. 1 (CC-BY-NC)
