iGEM 2019 — Team William_and_Mary

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# Team:William and Mary/Notebook

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Page Title

# Notebook

### Week 1: 190528 - 190602

* Set-up lab books and Google File Stream.
* Transformed quorum sensing parts into NEB-5a.
* Designed primers for quorum sensing parts to make them 3G compatible.
* Cloned pSB1C3.
* Starting to take deep learning course online, get some basics laid down before starting to clean up the database.
* STEM Community Day.

### Week 2: 190603 - 190609

* Cloned quorum sensing parts with W&M PAD.
* Tested projector set-up for pDAWN experiments.
* Designed primers to isolate and make pDAWN subunits 3G compatible (including AG43)
* Assembled and tested circuit WM19\_001 (tests Las QS system).
* Continue with deep learning course, starting to look for a python web crawler that could help us get all the information from other team’s outreach website
* Created outreach poster to present at Mid-Atlantic Synthetic Biology Symposium
* Presented past projects and outreach at Mid-Atlantic Synthetic Biology Symposium

### Week 3: 190610 - 190616

* Miniprepped pDAWN + AG43 control circuits.
* Designed and 3G assembled circuit WM19\_002.
* Plate reader experiment with circuit WM19\_002.
* PCRed and cloned now 3G compatible pDAWN subparts.
* Finished the modeling for Stanford Paper, tested their hypothesis and confirmed the validity of their conclusion.
* Starting to use their biofilm lithography method to form our own double ring experiment but dimension keeps getting dimensions wrong

### Week 4: 190617 - 190623

* Plate reader experiment on circuit WM19\_001.
* Gibsoned and transformed circuits WM19\_003-006.
* Plate reader experiment confirmed circuit WM19\_002 functionality.
* Blue-light optogenetic experiment on pDAWN + AG43, visualized with crystal violet.
* Transformed pDAWN + AG43 control circuits.
* Was able to get the double ring experiment dimensions correctly, but was not able to get the diffusion matrix to work as we expected, the number kept going higher and higher to infinity.
* Discussed with professor Patel, resolved this problem by changing the way that we convolve matrix while counting HSL concentration on one grid.
* Met with W&M admissions on ways to expand our outreach through their resources

### Week 5: 190624 - 1900630

+ Designed and 3G Assembled Tet-inducible AG43 circuit.
+ Fine tuning variables in blue-light experiments.
+ Completed iGEM safety form, inspiration form, IBC protocol, project description.
+ Tested pDAWN + AG43 control circuits.
+ Gibsoned and transformed circuits WM19\_007-012.
+ Continued testing of quorum sensing systems.
+ Co-transformed WM17\_399 and pDAWN AG43 for fluorescent biofilms.
+ Focused on utilizing machine learning method to clean up previous year’s outreach data, tried different types of RNNs, language mode, different scales of parameters. Without much luck because the best accuracy that we can get is 42% for project tags.
+ Presented to PLUS students at Navigating W&M event

### Week 6: 190701 - 190707

+ Designed primers to create quorum quenching parts based on HSL-binding domains of quorum sensing system.
+ Continued pDAWN + AG43 experiments.
+ Diagnostics of problematic circuits WM19\_007-008, WM19\_010-012.
+ Determined no reasonable way to do ultrasound induction as is inextricably linked to heat or oxygen stress.
+ 3G Assembled and miniprepped circuits WM19\_013-016.
+ Transformed circuits WM19\_13-16 into pDAWN + AG43 JS006.
+ Encountered some problems with our diffusion model: because of the way that we convolve the matrix, the run time is very long. But we are getting results for the double ring experiment, concentration of HSL does not meet our expectations.

### Week 7: 190708 - 190714

+ Plate reader experiment for quorum sensing systems.
+ Transformated quorum sensing systems WM19\_003, WM19\_005-006 into pDAWN+AG43 JS006.
+ Created of QUBES conference materials.
+ PCRed quorum quenching parts.
+ Plate reader ring-forming experiment with circuits WM19\_003-004.
+ Research for adhesin library.
+ Tested ATC-inducible AG43 circuits.
+ Gibsoned and transformed circuits WM19\_017-021.
+ Blue-light experiment with circuits WM19\_013-015.
+ Completed IBC protocol.
+ Prepped for PLUSS outreach event.
+ Blue-light experiment with media on agar.
+ Kept adjusting the parameters for the double ring experiment.

### Week 8: 190715 - 190721

+ Reviewed curli fibers; continued research on other adhesins.
+ Gibsoned and transformed circuit WM19\_022.
+ Reviewed curli and fap primers and geneblocks.
+ Blue-light experiment with circuit WM19\_022.
+ Exploring word2vec embedding method within scikit learn for our database
+ Read the turing paper, brainstorming ways of modeling the fast diffusing inhibitor and slow diffusing activator.
+ Participated in PLUS-S as teaching assistants and hosted 3 PLUS-S students for daily lab work
+ Presented our “Integrating Synthetic Biology and Big Data” poster and materials at the QUBES conference
+ Presented to Camp Launch students at Focusing on the Future

### Week 9: 190722 - 190728

+ Prepped for Mid-Atlantic Meet-up and presented there.
+ Plate reader experiment for quorum quenching.
+ Skype call with Dr. Joshi.
+ Prepped for outreach activity.
+ Colony PCRed off of Pseudomonas aeruginosa, Eschichriae coli, and Staphylococcus aureus for adhesins.
+ Distance-dependent quorum sensing experiment.
+ 3G assembled and transformed curli operon (divergent).
+ Designed and 3G assembled of arabinose-inducible AG43.
+ Gibsoned and transformed circuits WM19\_023-024.
+ Wrapping up with the turing model, looking through microbacteria paper and looking for ways to model microbacteria.
+ Hosted 30 Camp Launch for a day in the lab

### Week 10: 190729 - 190804

+ Troubleshooting fap, need to do site-directed mutagenesis.
+ Continued distance-dependent quorum sensing experiment.
+ 3G assembly of SaSuhB circuits.
+ Designed circuits WM19\_026-027.
+ Designed fap site-directed mutagenesis (SDM) primers (KLD).
+ Designed Turing pattern parts.
+ Outlined mycobacteria work.
+ Final adjustments for parameters according to some rough experimental data.

### Summer Break: 190805-190828

+ Wrote iGEM Summer Report (Project Description and Literature Review, Methodology)
+ Worked on OpenStax supplements
+ Reviewed and ordered SDM primers to remove illegal cutsite from fap from IDT
+ Reviewed and ordered Turing pattern parts from Twist and IDT
+ Ordered parts for alternative optogenetics circuit
+ Finalized curriculum

### Week 11: 190829-190910

+ Gibsoned and transformed circuits WM19\_030-035
+ Gibsoned and transformed Turing parts included in distribution kit
+ Made Turing parts from distribution kit 3G compatible
+ Gibsoned and transformed csgBAC and csgEFG geneblocks
+ Skype conference with University of Pittsburgh iGEM team
+ Diagnostics on circuits WM19\_030-035
+ Inoculated circuits WM19\_032-WM19\_034 for testing
+ Implemented cell collision, cell division, cell death in all models to increase their accuracy
+ Combined machine learning with web scraping database
+ Began advertising for Ladies in the Lab event

### Week 12: 190911-190914

+ Miniprepped csgBAC geneblock in Pad1C3
+ Retransformed circuits WM19\_030 and WM19\_031
+ Electrocompetent cell preparation
+ Ordered primers to add new sticky ends to some of the Turing pattern parts
+ Ring (quorum sensing) experiment by using blue light to place circuit WM19\_005 around circuit WM19\_006
+ Completed Safety Form

### Week 13: 190915-190923

+ Transformed circuits WM19\_023-024, WM19\_030-031
+ Transformed Dr. Joshi’s plasmid
+ Plate reader experiment with WM19\_023-024, WM19\_030-031, Joshi plasmid using congo red assay
+ Transformed circuits WM19\_023 and WM19\_031
+ Gibsoned transformed YF1/FixJ
+ Induced and imaged quorum sensing experiment under fluorescence microscope
+ Tested circuit WM19\_033 with congo red assay
+ Transformed circuits WM19\_032-WM19\_034
+ Skype call with Cornell iGEM team
+ Cloned fap operon
+ Site directed mutagenesis (SDM) with KLD and Gibson Assembly to remove the illegal cutsite in the fap operon and allow for 3G assembly
+ Assembled circuits WM19\_026 and WM19\_036
+ Gibsoned and transformed all Turing pattern parts
+ Changed sticky ends of WM19\_CD\_114, WM19\_CD\_116, WM19\_CD\_117 using designer primers

### Week 14: 190924-191009

+ Tested circuit WM19\_023 with congo red assay
+ Assembled stronger IPTG-inducible curli operon circuit and cI-repressed curli operon circuit
+ Co-transformed circuit WM19\_023 with pDAWN + AG43
+ Made competent mycobacteria cells and successfully transformed with shuttle vector
+ Designed geneblocks and primers for mycobacteria parts
+ Blue-light experiment for pDAWN + AG43 + WM19\_023
+ Ladies in the Lab event
+ Gibsoned and transformed WM19\_027
+ Plate reader experiment with WM19\_026-036, WM19\_027 with congo red assay
+ Imaged WM19\_026 in JS006 under fluorescence microscope
+ Researched mycobacteria parts
+ Ordered parts for collaboration with University of Pittsburgh
+ Worked on OpenStax supplement
+ Tested EL222/pBlind system
+ Tested circuits WM19\_033, WM19\_037, WM19\_040
+ Gibsoned circuits WM19\_029
+ Shared the finished curriculum with Virginia teams

### Week 15: 191010-191017

+ Assembled circuits WM19\_043-046, tested with congo red assay
+ Worked on Judging form
+ Ran statistical tests on experimental data
+ Tested WM19\_033, WM19\_037, WM19\_040-041 with congo red assay
+ Gibsoned and transformed mycobacteria parts
+ Used electroporation to transform competent mycobacterial cells
+ Imaged plates of mycobacteria under fluorescent microscope

### Week 16: 191016-191021

+ WIKI WORK!!!!

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* Contact us at igem@wm.edu

Sponsors

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2019 William & Mary iGEM


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# Team:William and Mary/Experiments

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Page Title

# Experiment

## Protocols

#### We have uploaded all of our protocols for lab tasks as a [zip file.](https://static.igem.org/mediawiki/2019/3/32/T--William_and_Mary--protocols.zip)

#### A list of contents is found here:

* 3G Assembly (contains instructions for Golden Gate and PCR amplification of Golden Gate product. Additional protocols must be used for subsequent gel extraction of PCR product and Gibson of transcriptional units into vectors)
* Annealing 3G Adapters (how to anneal UNS sequences when ordered as single-stranded oligos)
* Biofilm Lithography (follows protocol laid out in 2018 PNAS paper by Jin & Riedel-Kruse)
* Centrifuge (how to properly balance and operate the centrifuge)
* Colony PCR (assess colonies for successful transformation)
* DpnI Digestion (remove methylated template DNA after PCR)
* Electroporation of M. smegmatis (for competent M. smegmatis cells)
* Filter Sterilization
* Gel Electrophoresis
* Gel Extraction (follows 3G Assembly Protocol. Also aids in isolation of desired DNA when multiple bands are present on gel electrophoresis gels)
* Gene Block Resuspension
* Gibson Assembly (final step for plasmid assembly via 3G DNA Assembly. Combines transcriptional units into a backbone utilizing UNS overlaps. Gibson Assembly is also used to place parts with “Pad” adapter sequences into William and Mary’s “Pad 1C3,” a cloning backbone)
* Inoculation for Miniprep
* Making Competent Cells (makes JS006 experimental cells competent. Competent DH5-alpha cells are bought from NEB)
* Making LB Agar Plates
* Midiprep
* Miniprep
* Nanodrop (proper operation and cleaning of Nanodrop used to quantify DNA concentration and purity)
* PCR Purification
* Plate Reader (how to properly operate and collect data using our Synergy plate reader)
* Resuspension of primers (received in dried form)
* Standard PCR
* Transformation

## Methods

### Induction and Assessment of Adhesins

To assess pDawn-AG43, we strictly followed the methods of "Biofilm Lithography enables high-resolution cell patterning via optogenetic adhesin expression" and the related Jove information video (Jin & Riddle-Kruse, 2018). Though we used a Magnasonic LED Pico Projector to project blue light, rather than the original Ivation Pro4 projector used by Jin and Riedel-Kruse, we followed other instructions regarding inoculations, dilutions, and blue slide preparation on Microsoft Powerpoint. These methods were summarized in a protocol we drafted called “Biofilm Lithography.” Blue-light induction of both pBlind-curli containing circuits followed this protocol, as well. This protocol, which provides instructions for crystal violet staining of AG43, is contained within the zip file.

To assess amyloid fiber production, we utilized the Congo Red spin-down assay designed by Harvard iGEM’s 2017 team, OptiPoly. 3 mL LB broth was inoculated with 3 uL colony solution (one colony in 10 uL nuclease-free water) and the culture was allowed to grow overnight. The next day, cells were stained via a 0.015% Congo Red solution, then centrifuged and pelleted. If cells had expressed curli fibers, the Congo Red stain would bind the fibers and end up in the cell pellet, resulting in a lighter supernatant than samples lacking curli fibers. The color of the supernatant, along with the OD600 of the original culture sample, was quantified using a plate reader. However, difference in pellet color was often apparent to the naked eye. As well, aggregation in the bottom of inoculated glass tubes was often observed prior to Congo Red staining. See the full, step-by-step protocol [here.](https://2017.igem.org/Team%3AHarvard/Protocols)

![](https://static.igem.org/mediawiki/2019/8/8c/T--William_and_Mary--23aggcomp.jpg)

Visible differences prior to quantitative data collection: visible aggregation on the bottom of glass culture tubes with induced samples (left photo, right tube). Uninduced samples show no such aggregation (left photo, left tube). Before plate reader analysis, Congo Red results are visible (right photo). The induced sample (rightmost 1.7 mL centrifuge tube) has a redder pellet than the uninduced sample (middle 1.7 mL centrifuge tube), which closely resembles the untransformed JS006 negative control (leftmost 1.7 mL centrifuge tube).

To assess inducible circuits, two cultures were inoculated per colony solution, ensuring that the cells within both tubes were genetically identical. One culture was induced while the other was not. To assess constitutive circuits such as J23107-curli, equal numbers of experimental and negative control (untransformed JS006) tubes were inoculated to enable paired t-tests.

IPTG induction for curli fibers was eventually accomplished using 10 uL 1M IPTG/3 mL, ~3.3 mM final concentration (see Results page for results of preliminary experiments with various inducer concentrations). IPTG induction of SaSuhB was accomplished with final IPTG concentration of 10 mM, while IPTG induction of fap was accomplished with a final concentration of ~24.4mM. Untransformed JS006 were inoculated as negative controls for IPTG-inducible curli experiments. pBbB8K-csgBACEFG, a verified synthetic curli operon from Dr. Neel S. Joshi at Harvard's Wyss Institute, served as a "positive control" to exemplify what robust curli expression should look like. See the results page for more information.

### Quorum Sensing and Distance-Dependent Patterning

To assess our quorum sensing circuits’ ability to establish distance-dependent patterning, we devised a “ring experiment” utilizing the blue light-induced pDawn system. First, we created stocks of competent JS006 cells with the pDawn-AG43 plasmid. Then, we cotransformed our quorum sensing circuits into these competent cells, so that both sender and receiver strains had the pDawn-AG43 plasmid. Since our cells were cotransformed with this plasmid, we could use blue light biofilm lithography to place a small circle of sender strain cells on a petri dish. The next day, we aspirated the liquid media (leaving the sender strain biofilm on the plate) and replaced it with M63 inoculated with receiver strain bacteria. We again used biofilm lithography to create a biofilm, this time locating receiver strain bacteria in a ring surrounding the circular sender strain biofilm. After overnight placement of this ring biofilm, we induced the sender strain bacteria using a thin lawn of M9 + IPTG (1.5 mL total volume with 10 mM final concentration of IPTG; 15 uL 1M IPTG and 1450 uL M9). We waited two hours, allowing time for induced sender strain bacteria to produce HSL, for this HSL to diffuse outward into the ring of receiver strain bacteria, and for the ring of receiver strain bacteria to respond to the HSL and produce mScarlet. Results were visualized using a dissection scope.

Theoretically, the diffusion of HSL from the sender strain results in distance-dependent patterning. Fluorescence should be strongest in a ring surrounding the circular sender biofilm, and this ring of fluorescence should fade outwards in a gradient (since HSL, the inducer of this fluorescence, decreases in concentration as it diffuses away from the sender circle). Though presence of both sender and receiver strains was confirmed, and induction of the receiver ring by the sender was visualized, the expected gradient of fluorescence was not observed (see results page). The entire outer ring appeared equally fluorescent, which we attribute to over-induction of the sender strain by IPTG (and thus over induction of the receiver strain by HSL). Following this experiment, we realized that our biofilm was larger in size than that simulated by the math modelling team. We asked them to expand the scale of their model, as well as model different IPTG concentrations and incubation times. Future experiments will follow their suggestions for ideal induction of the sender strain.

Though our final ring experiment proved inconclusive, “benchmark” plate reader experiments with prototype circuits allowed us to assess the functionality of our quorum sensing circuits. For example, plate reader data from prototype circuits confirmed that 1) sender strain bacteria are successfully induced by IPTG 2) receiver strain bacteria are successfully induced by HSL 3) sender strain bacteria can successfully induce receiver strain bacteria when induced by IPTG. To verify conclusion 1, colonies of a prototype sender circuit (IPTG induction results in mScarlet production) were exposed to IPTG and red fluorescence was monitored over time. To verify conclusion 2, receiver strain were directly induced with HSL and fluorescence was monitored over time. To verify conclusion 3, both sender and receiver cells were grown together in one well. Fluorescence was measured over time after addition of IPTG. Fluorescence in the third scenario was further visualized by microscopy.

As well, prototype and final quorum-sensing circuits were sequence confirmed by Sanger sequencing on both strands by Epoch Life Sciences.

## References

#### [1] Jin, X., & Riedel-Kruse, I. H. (2018). Biofilm lithography enables high-resolution cell patterning via optogenetic adhesin expression. PNAS 115, 3698-3703. doi: 10.1073/pnas.1720676115

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# Team:William and Mary/Results

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  + [Overview](https://2019.igem.org/Team%3AWilliam_and_Mary/Overview)
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  + [Public Engagement](https://2019.igem.org/Team%3AWilliam_and_Mary/Public_Engagement)
  + [Education](https://2019.igem.org/Team%3AWilliam_and_Mary/Education)
* [Model](https://2019.igem.org/Team%3AWilliam_and_Mary/Model)
* [Database](https://2019.igem.org/Team%3AWilliam_and_Mary/Software)

Results

# Results

# Adhesin Library

## Functional Amyloid in Pseudomonas (FAP)

BBa\_K3059640 (strong constitutive fap operon) and BBa\_K3059641 (weak constitutive fap operon) were characterized with OD600 and congo red assays 20 hours after inoculation. Using a two-tailed paired T-test, a statistically significance difference was found between the absorbance of congo red stained JS006 cells and BBa\_K3059640 (p = 0.00017, n = 5), JS006 and BBa\_K3059641 (p = 1.59 x 10-5, n = 5) and BBa\_K3059640 and BBa\_K3059641 (p = 0.0125, n = 5). BBa\_K3059641 on average retained slightly more congo red dye than BBa\_K3059640. One explanation for the results between the strong and weak circuits may be the high production rate of a large part like BBa\_K3059637 (3G compatible fap operon) results in slower growth, allowing cells with lower levels of expression to double faster and produce overall more fiber. However, when considering the average OD600 for the two groups, BBa\_K3059640 actually had a higher average absorbance than its weaker counterpart BBa\_K3059641, indicating more growth. Overall, the hypothesis that these circuits would express more amyloid (fap) nanofibers, leading to more congo red binding, is supported.

![](https://static.igem.org/mediawiki/2019/0/05/T--William_and_Mary--26and27pict.png)

![](https://static.igem.org/mediawiki/2019/a/a3/T--William_and_Mary--26and36chart600.png)

![](https://static.igem.org/mediawiki/2019/e/ef/T--William_and_Mary--26and36chart490.png)

Decrease in A490 of BBa\_K3059640 and BBa\_K3059641

BBa\_K3059634 (IPTG-inducible fap operon) was characterized with OD600 and congo red assays 20 hours after inoculation. There were four test groups: induced BBa\_K3059634, uninduced BBa\_K3059634, induced JS006, and uninduced JS006. Using a two-tailed paired T-test, a statistically significant difference was found between the absorbance of induced BBa\_K3059634 and both JS006 groups (p = 0.0039, n = 10; p = 0.0011, n = 10), as well as between uninduced BBa\_K3059634 and both JS006 groups (p = 0.0010, n = 10; p = 0.0013, n = 10). There was no significant difference between the induced and uninduced BBa\_K3059634 groups (p = 0.1937, n = 10). This seems to indicate leaky repression of pLac, possibly since LacI expression is under the control of the weak promoter J23107. As this part was only functionally confirmed, it is also possible that there is a mutation in pLac or other regulatory gene of the fap operon.

![](https://static.igem.org/mediawiki/2019/5/5c/T--William_and_Mary--27spinimage.png)

Induced BBa\_K3059634

![](https://static.igem.org/mediawiki/2019/a/a2/T--William_and_Mary--x27spinimage.png)

Uninduced BBa\_K3059634

![](https://static.igem.org/mediawiki/2019/0/05/T--William_and_Mary--js006spin.png)

Induced JS006

![](https://static.igem.org/mediawiki/2019/b/ba/T--William_and_Mary--xjs006spin.png)

Uninduced JS006

![](https://static.igem.org/mediawiki/2019/e/e6/T--William_and_Mary--27chart600.png)

OD600 of induced and uninduced BBa\_K3059634.

![](https://static.igem.org/mediawiki/2019/b/b6/T--William_and_Mary--27chart490.png)

Decrease in A490 of induced and uninduced BBa\_K3059634

## Synthetic Curli Operon

### We assembled and characterized four curli fiber-producing circuits:

#### [1] WM19\_023, or “circuit 23”: IPTG-inducible curli

#### [2] WM19\_024, or “circuit 24”: Weak constitutive curli

#### [3] WM19\_045, or “circuit 45”: pBlind + curli

#### [4] WM19\_046, or “circuit 46”: pBlind + AG43 + curli

## Curli Amyloid Fibers

Of the four curli fiber circuits we constructed, WM19\_023 and WM19\_024 were constructed first. Before experiments began, we performed a diagnostic restriction digest with enzyme EcoRI. Though Type IIS-compatible and thus legal, the native curli operon contains an EcoRI cutsite, so two cuts were expected: EcoRI within the Biobrick prefix and EcoRI within csgE (internal cut). Analyzing the digests with these cutsites in mind, all minipreps of circuit 23 and 24 appeared correct.

![](https://static.igem.org/mediawiki/2019/a/af/T--William_and_Mary--23dd.jpeg)

restriction digest on circuit 23. Expected bands at ~2 kb at ~5 kb appear.

![](https://static.igem.org/mediawiki/2019/2/28/T--William_and_Mary--24dd.jpeg)

restriction digest on circuit 24. Expected bands at ~1.9 kb and ~3.5 kb appear.

## WM19\_023

Preliminary experiments with circuit 23 aimed to determine a reasonable amount of IPTG to add for curli fiber expression. We initially followed the Harvard OptiPoly protocol, hoping that the instructions for arabinose induction of pBbB8K-csgBACEFG would work similarly for IPTG induction of WM19\_023. However, 0.75 uL of 1M IPTG into 3 mL LB caused no observable curli fiber expression or statistically significant difference in supernatant color. A second experiment with 3 uL 1M IPTG into 3 mL LB (~1 mM final concentration) proved similarly ineffective. To verify that the circuit was at all functional, we induced with 30 uL 1M IPTG into 3 mL LB (~10 mM final concentration), and finally saw results (both to the naked eye and in plate reader data). Though we needed more replicates to draw any conclusions regarding the effectiveness of circuit 23, this experiment provided us with a starting point for IPTG induction.

![](https://static.igem.org/mediawiki/2019/b/b6/T--William_and_Mary--23w30.jpg)

When induced with 30 uL 1M IPTG, cell pellets were visibly redder than uninduced counterparts. The redness of these pellets should not be compared to that of pellets in future experiments, however; 150 uL of 0.015% Congo Red solution was mistakenly used instead of 100 uL.

Believing the full 30 uL to be excessive, we tested induction with 10, 20, and 30 uL of 1M IPTG. From visual inspection and plate reader data, we determined that no significant difference in curli production/supernatant color occurred past 10 uL 1M IPTG, and quickly devised a follow-up experiment to confirm the effectiveness of 10 uL. This experiment showed that induced samples consistently formed visible aggregates in glass culture tubes overnight, and consistently produced redder pellets/clearer supernatants (confirmed by plate reader) after staining with Congo Red solution.

![](https://static.igem.org/mediawiki/2019/e/e3/T--William_and_Mary--23pellets.png)

Pellets visible on the bottom of glass culture tubes with induced samples. No such aggregation was visible for negative control samples of untransformed JS006.

![](https://static.igem.org/mediawiki/2019/1/19/T--William_and_Mary--23N0.jpg)

Uninduced cultures, like the one above, showed no aggregation after overnight growth.

![](https://static.igem.org/mediawiki/2019/d/d6/T--William_and_Mary--23showcasefull.jpg)

From left to right: WM19\_023 1C3 #1 uninduced, WM19\_023 1C3 #1 induced, WM19\_023 1C3 #2 uninduced, WM19\_023 1C3 #2 induced, WM19\_023 1C3 #3 uninduced, WM19\_023 1C3 #3 induced, WM19\_023 1C3 #4 uninduced, WM19\_023 1C3 #4 induced, untransformed JS006 #1, untransformed JS006 #2, uninduced pBbB8K-csgBACEFG, induced pBbB8K-csgBACEFG. pBbB8K-csgBACEFG is a gift from Dr. Neel S. Joshi at Harvard’s Wyss Institute. We discuss this plasmid later.

As seen above, for each pair of samples, the induced/right of the two samples has a redder pellet. Uninduced samples (the left of each pair) resemble the negative control, untransformed JS006. A student’s t-test (n=4, p=0.0196) found statistically-significant difference between uninduced and induced samples with a confidence level of 98.0%.

![](https://static.igem.org/mediawiki/2019/7/76/T--William_and_Mary--234sampleR.jpg)

Box and whisker graph comparing Congo Red spin down assay results for uninduced and induced samples. For each sample, A490 of supernatant (provided by plate reader) was subtracted from A490 of a positive control (pure 1X PBS mixed with 100 uL 0.015% Congo Red solution). Samples that produced curli fibers bound more Congo Red dye, resulting in a greater difference between pure PBS/Congo Red solution and supernatant. As seen in the graph above, induced samples bound more Congo Red dye, resulting in a greater difference between supernatant A490 and control than uninduced samples. Blue lines represent averages, while red dots represent individual samples.

However, only four colonies were tested in this preliminary assessment. A final experiment tested ten colonies, producing 22 tubes total (ten induced, ten uninduced, two untransformed/negative control). To ensure that uninduced/induced sample pairs contained genetically identical bacteria, each inoculated with the same colony solution.

Again, results were apparent even before qualitative assessments: aggregates formed overnight in induced cultures, and cell pellets for induced samples were redder than their counterparts. Plate reader absorption measurements at 490 nm showed a consistent decrease in supernatant redness (Congo Red bound in the cell pellet decreases supernatant redness) from uninduced to induced samples. This difference was verified by a student’s t-test as statistically significant (n=10, p=0.00298) with a confidence value of 99.7%.

![](https://static.igem.org/mediawiki/2019/8/88/T--William_and_Mary--2310sample.jpg)

Box and whisker plot formatted identically to the one above. Once again, induced samples bound more Congo Red dye, resulting in a greater difference between supernatant A490 and control than uninduced samples. Blue lines represent averages, while red dots represent individual samples.

During these experiments, we also tested pBbB8K-csgBACEFG, a synthetic curli plasmid from Neel S. Joshi. This plasmid showed dramatic curli expression when induced, compared to our IPTG-inducible circuit. However, cells with pBbB8K showed impressive curli production even when uninduced. Uninduced samples of the arabinose-inducible circuit resembled the induced samples of our IPTG-inducible circuits, rather than the negative control (untransformed JS006).

![](https://static.igem.org/mediawiki/2019/7/75/T--William_and_Mary--comppBbB8K.jpg)

From left to right: untransformed JS006, uninduced IPTG-inducible curli circuit, induced IPTG-inducible curli circuit, uninduced pBbB8K-csgBACEFG, induced csgBACEFG. The uninduced sample of pBbB8K-csgBACEFG resembled the induced sample of our IPTG-inducible circuit, rather than the negative control. The data suggests that, though our circuit maintains weaker expression of curli fibers, expression is less leaky.

### WM19\_024

WM19\_024 placed our synthetic curli operon under medium-weak constitutive promoter J23107. No visible pellets formed in the glass culture tubes during overnight growth, and no difference in redness was observed between control and experimental pellets after Congo Red staining. Furthermore, the average A490 measurement for experimental samples’ supernatants was slightly higher than the average for control circuits (if curli fibers were expressed, the supernatant should be lighter, resulting in a lower A490 measurement). Future constitutive circuits may require a stronger promoter such as J23100.

### WM19\_045

WM19\_024 places our synthetic curli operon under the promoter pBlind, which is activated by blue-light sensitive transcription factor EL222 (Jayaraman et al., 2016). Both pBlind and EL222 were obtained through DNA synthesis, and sequence-confirmed in multiple circuits.

Though considered an optogenetic circuit, pBlind-EL22 had resulted in constitutive gene expression in circuit 46, which was miniprepped before circuit 45. With this in mind, we tested the function of circuit 45 when exposed to ambient light as well as blue light.

Our first experiment to test WM19\_045 did not involve blue light specifically. Five experimental tubes were exposed to ambient light while growing in the shaking incubator overnight, while experimental tubes were covered with foil (foiled and unfoiled sample pairs were inoculated using the same colony solution, allowing for direct comparison). JS006 cultures were grown as a negative control. Some experimental tubes, both foiled and unfoiled, showed small aggregates at the bottom. However, no obvious differences in pellet color were visible to the naked eye.

![](https://static.igem.org/mediawiki/2019/4/44/T--William_and_Mary--451A.jpg)
![](https://static.igem.org/mediawiki/2019/8/81/T--William_and_Mary--451F.jpg)

#### A sample that had been exposed to ambient light (left) as well as a sample that had been covered with foil (right). Both have small aggregates at the bottom.

The average A490 value for each group reflected expectations: average A490 was the lowest for experimental samples exposed to ambient light, followed by the average for foiled samples, followed by the average for the negative controls. However, these averages were hardly different (0.2438, 0.2440, 0.2482), and no statistical significant difference was found between ambient light and foiled samples (n=5, calculated t=-0.0673). Statistical significance was found between ambient light and control samples (n=5, calculated t=-2.475, 2.475 > 2.132) but only with a confidence level of 90%.

A second experiment exposed samples of circuit 45 to blue light. Two six-well plates were inoculated with three colonies of circuit 45 and three control (untransformed JS006) colonies. One plate was projected with blue light while the other was grown within a dark box. Both plates were grown in a stationary incubator and filled with M63 media rather than LB broth.

A clear difference between experimental and control samples was visible the next day; aggregation was observed within experimental wells whereas the turbidity within control wells appeared more “smooth.” This trend was apparent for both the plate grown under blue light and the plate grown in darkness.

![](https://static.igem.org/mediawiki/2019/2/2b/T--William_and_Mary--45bluelight.jpg)
![](https://static.igem.org/mediawiki/2019/b/be/T--William_and_Mary--45dark.jpg)

#### For both the plate exposed to blue light (left image) and the plate grown in the dark (right image), experimental wells (left of two wells) showed aggregation whereas control wells (right of two wells) were turbid but uniformly so. Furthermore, experimental pellets (both those grown in darkness and those exposed to blue light) stained a darker red than negative control pellets.

![](https://static.igem.org/mediawiki/2019/b/bc/T--William_and_Mary--45BLCR.jpg)

#### Two stained samples of circuit 45 between two negative control samples. Note the redder pellets of the middle samples. Both are similarly red, redder than the negative controls, although one was exposed to blue light and the other was grown in darkness.

No statistically significant difference was found between WM19\_045 samples grown in the dark and samples exposed to blue light. Though curli expression (and thus A490) appeared to decrease from experimental samples to negative control samples, it was difficult to verify this relationship statistically with only n=3. The inability of the projector to cover many samples with intense blue light prevented the use of large sample sizes. Though future experiments were designed, such experiments were unfeasible with the time constraints.

Our available data suggests that, if pBlind is truly light-sensitive, the level of induction by blue light is somewhat weak (especially compared to the pDawn system). Instead, pBlind seems to express downstream genes constitutively, even moreso than our constitutive circuit J23107-curli (WM19\_045).

### WM19\_046

Like WM19\_045, WM19\_046 utilizes the pBlind-EL222 system. However, circuit 46 includes both AG43 and curli fibers. As with circuit 45, pBlind-EL222 appears to result in constitutive, uninduced expression. Inoculations for miniprep, though covered completely in foil and grown in a shaking incubator, exhibited massive aggregates at the bottom of glass culture tubes and minimal cells in the LB supernatant.

![](https://static.igem.org/mediawiki/2019/d/d3/T--William_and_Mary--46forMP.jpg)

#### Aggregates at the bottom of inoculations for miniprep, resulting in clearer LB broth supernatant.

![](https://static.igem.org/mediawiki/2019/e/e8/T--William_and_Mary--46A.jpg)
![](https://static.igem.org/mediawiki/2019/e/ee/T--William_and_Mary--46FJS.jpg)

This behavior persisted in JS006 samples inoculated for experiment. Though no statistically significant difference was found between foiled and unfoiled experimental samples, a statistically significant difference was found between unfoiled experimental samples and the negative control (n=5, calculated t=-3.8069, 3.8069 > 3.747) with a confidence interval of 98%. If one assumes that light plays little to no role in adhesin production and combines foiled and unfoiled groups, the confidence interval rises to 99.9% (n=10, p=0.000389).

![](https://static.igem.org/mediawiki/2019/7/77/T--William_and_Mary--46graph.jpg)

#### Box and whisker graph comparing circuit 46 samples to untransformed JS006, the negative control. Samples expressing curli fibers bound more Congo Red dye, resulting in lighter supernatants and lower A490 values. As shown in the graph above, circuit 46 samples had generally lighter supernatants than JS006 samples. Blue lines represent averages while red circles represent individual samples.

We also completed a blue light experiment identical to the one used for circuit 45. However, instead of performing a Congo Red assay on the cultures, the wells were crystal violet stained. Before crystal violet staining, aggregation was visible in experimental wells (as opposed to uniformly turbid negative control wells). Unfortunately, aspirating the media and subsequently washing with 1X PBS removed most of the aggregation produced by experimental samples. This suggests that AG43 and curli fibers were expressed constitutively by cells floating freely within the liquid media. Had the cells expressed AG43 as a result of adsorbing onto the region of the plate exposed to blue light (as proposed by the original PNAS paper by Jin and Riedel-Kruse), a purple film would have been observed after aspiration and washing. Instead, it appears that most of the adhesin was suspended in the liquid media.

Though optogenetics was the intended use for circuits 45 and 46, we now have constitutive circuits to replace the ineffective circuit 24.

## SaSuhB

### IPTG-Inducible Circuits

To assess amyloid fiber formation for each of the SaSuhB circuits, a congo red assay was performed for each of the circuits. Colonies of 033 and 040 were picked, and inoculated into a colony solution. Half of the colony solution was added to 4mL of LB + chloramphenicol, and the other half of the colony solution was added to 4mL of LB + chloramphenicol + 10mM IPTG to induce expression of SaSuhB. After being grown for 18 hours, the solutions were Congo Red stained using our standardized protocol.

![](https://static.igem.org/mediawiki/2019/4/45/T--William_and_Mary--SaSuhB_033.png)
![](https://static.igem.org/mediawiki/2019/2/27/T--William_and_Mary--SaSuhB_CR.jpg)

As can be seen in the graph above, the induced circuit 033 has a significantly higher decrease in A490 as compared to uninduced circuit 033 (p = 0.013678551, n = 4). This result is confirmed qualitatively by looking at the congo red spin down solutions. The tube on the left is uninduced, while the tube on the right is induced with 10mM IPTG.

![](https://static.igem.org/mediawiki/2019/d/d9/T--William_and_Mary--SaSuhB_040.png)

Circuit 040 was grown for 18 hours as well. As can be seen, both uninduced circuit 040 ( p=2.08 X 10^-06, n = 8) , and induced circuit 040(p = 0.0003, n = 8) have a significantly higher decrease in A490 as compared to uninduced circuit 033. Furthermore, the induced circuit 040 also has a significantly higher decrease in A490 as compared to uninduced circuit 040 (p = 0.038, n = 8).

### EL222-Circuits

Circuit 039 was inoculated directly into 4mL of LB + chloramphenicol. To induce expression of circuit 039, we found that ambient lighting was sufficient. After being grown overnight at 37C with 250rpm shaking, visible SaSuhB fibers were isolated from several of the culture tubes.

![](https://static.igem.org/mediawiki/2019/1/1e/T--William_and_Mary--SaSuhB_Slide.jpg)
![](https://static.igem.org/mediawiki/2019/b/b7/T--William_and_Mary--SaSuhB_Fluorescent.png)

Qualitatively, we found that the fibers were incredibly sticky, and we had difficulty removing the fibers from the side of the glass culture tubes if they touched the sides.

### Engineering Mycobacterium

#### Result Summary:

* 8 Function-confirmed Mycobacterium smegmatis compatible 3G parts (3 promoters, 2 RBSs, 2 terminators)
* 1 3G compatible shuttle vector with dual origin of replication for both E. coli and M. smegmatis
* 2 Constitutive gene expression circuits (mScarlet-I for testing)
* Validated Electroporation and Electrocompetent cell protocol

#### Electroporation Protocol Validation

Mycobacterium smegmatis is difficult to transform using traditional chemically-competent method. Thus, we adapted the electrocompetent cell protocol from Dr. Broussard lab (Broussard, 2009)and electroporation protocol from Cirillo et al. (1993) and Goude et al. (2008). To test out these protocols, we electroporated pSUM36 plasmid with gfp inserted (acquired from addgene). We imaged the transformed M. smegmatis under inverted fluorescence microscope. The validation test showed a positive result, which confirmed that the adapted protocols work well for our strain of Mycobacteria.

![](https://static.igem.org/mediawiki/2019/5/51/T--William_and_Mary--100X_pSUM-LacZ-gfp_M.smeg_FITC.jpg)

Magnification: 1000X. Filter: FITC

![](https://static.igem.org/mediawiki/2019/e/e2/T--William_and_Mary--1100X_pSUM-LacZ-gfp_M.smeg_TRANS.jpg)

Magnification: 1000X. Filter: Transmission

#### Test Circuits Characterization

In order to confirm that BBa\_K3059423 and BBa\_K3059424 function as we expected, we imaged Mycobacterium smegmatis transformed with these two constructs under fluorescence microscope.

![](https://static.igem.org/mediawiki/2019/3/3e/T--William_and_Mary--100X_BBa_K3059423_M.smeg_RBG-TRITC.jpg)

![](https://static.igem.org/mediawiki/2019/b/b5/T--William_and_Mary--100X_BBa_K3059423_M.smeg_TRANS.jpg)

![](https://static.igem.org/mediawiki/2019/f/f2/T--William_and_Mary--100X_BBa_K3059423_M.smeg_RBG.jpg)

M. smegmatis transformed with BBa\_K3059423. Magnification: 1000X. Filter: RGB-TRITC -> Transmission -> RGB

![](https://static.igem.org/mediawiki/2019/3/39/T--William_and_Mary--100X_BBa_K3059424_M.smeg_RBG-TRITC.jpg)

![](https://static.igem.org/mediawiki/2019/7/76/T--William_and_Mary--100X_BBa_K3059424_M.smeg_TRANS.jpg)

![](https://static.igem.org/mediawiki/2019/5/56/T--William_and_Mary--100X_BBa_K3059424_M.smeg_RBG.jpg)

M. smegmatis transformed with BBa\_K3059424. Magnification: 1000X. Filter: RGB-TRITC -> Transmission -> RGB

![](https://static.igem.org/mediawiki/2019/8/80/T--William_and_Mary--100X_Untransformed_M.smeg_RBG-TRITC.jpg)

![](https://static.igem.org/mediawiki/2019/9/97/T--William_and_Mary--100X_Untransformed_M.smeg_TRANS.jpg)

![](https://static.igem.org/mediawiki/2019/6/62/T--William_and_Mary--100X_Untransformed_M.smeg_RBG.jpg)

Untransformed M. smegmatis. Magnification: 1000X. Filter: RGB-TRITC -> Transmission -> RGB

To further quantify the gene expression level, we measured the optical density and fluorescence intensity for both transformed strains with plate reader experiments. Experimental data with BBa\_K3059423 and BBa\_K3059424 is plotted in the following graph. BBa\_K3059423 showed a 10-fold to 50-fold increase in F.I., while BBa\_K3059424 increased F.I. by up to 1500X fold.

![](https://static.igem.org/mediawiki/2019/4/49/T--William_and_Mary--M03%2BM04.png)

Fluorescence intensity normalized with respect to OD600 plotted on a log scale. Each dot represents a distinct biological replicate (colonies). Negative control measures untransformed, nonfluorescent M.smeg mc2155 while blank measures 7H9 media with no inoculation.

## Patterning

## Engineered Quorum Sensing

![](https://static.igem.org/mediawiki/2019/9/96/T--William_and_Mary--Characterization.png)