iGEM 2021 — Team Austin_UTexas

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# Team:Austin UTexas/Notebook

[Template:Austin UTexas/NotebookTemplate](/wiki/index.php?title=Template:Austin_UTexas/NotebookTemplate&action=edit&redlink=1 "Template:Austin UTexas/NotebookTemplate (page does not exist)")

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

### Inspiration

You can see what others teams have done to organize their notes:

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

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# Team:Austin UTexas/Engineering

# Engineering Success

### **Summary of Design Cycle**

![](https://static.igem.org/mediawiki/2021/f/f0/T--Austin_UTexas--DesignBill.jpg)

**Scheme of overall project design** After designing and assembling our final OCTOphage, the phage will be able to infect a broad range of bacterial hosts and insert our enzymes of interest. The genes for plastic degradation and oil emulsification will be expressed. After the phage lyses the host cell, the enzymes will be released into the surrounding water to clean up plastic and oil pollution. Figure created by team member Bill Tang

   The end goal of our project was to use P1 to create a phage that could insert plastic degradation or oil emulsification genes into a broad range of hosts, also known as OCTOphage. P1 was the ideal choice of phage due to its broad host range allowing it to inject our engineered phagemid into a wide variety of marine bacteria. In addition, P1 exhibits a relatively unique mechanism of randomly packaging the bacterial plasmid from its host in its capsid. Furthermore, there's a specific packaging sequence called *pacA* that increases the packaging rate of non-phage plasmids.

   In order to engineer the P1 phage's phagemid we had to go through multiple rounds of the design cycle. We first used Golden Gate Assembly to assemble our genes of interest into two first stage assemblies. One for our plastic degradation genes and one for our oil emulsification genes. We designed the two types of genes into separate assemblies because in our proposed implementation they would have different environments of application.

   The plastic degradation genes we used were PETase and MHETase; and the oil emulsification genes we tested were Ranaspumin-2 (Rsn-2), iLOV-Ranaspumin-2 (iLOV-Rsn2) and Latherin. We also used *pacA* to package the phagemid at an increased rate, so that more phages can package our genes of interest. For each of these genes, we created various Golden Gate part plasmids which we then assembled together to create our phagemid, or stage one assembly.

   We then transformed the phagemids, or stage one assemblies, into NEB5α *E. coli*. After, we infected each strain with P1 to allow P1 to package and take up our assemblies, creating OCTOphage. Then we infected wild-type of MG1655 *E. coli* with our OCTOphages to create a phage lysate and test that lysate on plastic degradation and oil emulsification assays respectively, determining if the OCTOphages would inject the plastic degrading and oil emulsifying genes into the wildtype *E. coli*, causing them to produce the corresponding enzymes. Our negative controls for these assays were phage lysates of wild-type MG1655 *E. coli*. Our positive controls were purified enzymes from the original Addgene PET plasmids we ordered ([pET21b(+)-Is-PETase](https://www.addgene.org/112202/), [pET28-Rsn2](https://www.addgene.org/61686/), [pET28-iLOV-Rsn2](https://www.addgene.org/61687/)) that were first over expressed using IPTG induction, and then purified. We transformed C41 and LysY/Iq *E. coli* with the Addgene plasmids of our plastic and oil degradation genes and then used His-tag purification to purify our enzymes.

### Build

![](https://static.igem.org/mediawiki/2021/2/2e/T--Austin_UTexas--Phagemid.png)

**Scheme of Phagemid Golden Gate Assembly** The figure above shows the intended final phagemid to be made using Golden Gate Assembly (GGA).

   The first step in creating OCTOphage was to create our part plasmid assemblies for plastic degradation and surfactant genes. For our plastic degradation scheme, we used PETase and MHETase as a joint coding sequence. For our surfactants we created three different coding sequences for Latherin, Ranaspumin-2 and iLOV-Rsn, our candidate enzymes. We also created a part for *pacA*, the packaging sequence used by P1 bacteriophage. For each part, we create primers on Benchling to add the correct GGA part overhangs. For each of the template sequences, we ordered PET plasmids off of Addgene and amplifed our gene of interest with PCR, adding Golden Gate overhangs int the process. These overhangs were used to assembly the coding sequences into entry vectors. These plasmids were transformed into NEB5 alpha chemically competent cells and checked for successful Golden Gate/transformation using selective marker CAM resistance (successful transformation) and screenable marker GFP (successful assembly). Next, we picked colonies, grew liquid cultures, mini prepped, sequenced to verify that no mutation were present. The same procedure was used for the parts 1, 2, 4, 5, and 8 which were obtained from the bee tool kit (BTK) and yeast tool kit (YTK). After all Golden Gate parts were successfully created, two stage 1 assemblies were created for plastic degradation and surfactants enzymes. To check for successful Golden Gate/transformation we used a selective marker, CRB resistance (successful transformation), and a screenable marker, RFP (successful Golden Gate).

### Test

![](https://static.igem.org/mediawiki/2021/3/37/T--Austin_UTexas--ES_fig2.jpg)

**Creating OCTOphage** Using P1 transfection mechanisms and sequence packaging, P1 incorporates Golden Gate First Stage Assembly plasmids from the transformed host. Upon lysis, OCTOphage is successfully assembled with the desired PETase and Ranaspumin-2 genes. OCTOphage is then ready to deliver these genes to infect target host strains. Figure created by team member Bill Tang

   After we created the first stage assemblies for surfactant and plastic degradation genes with the P1 packaging sequence, we transformed *E. coli* with these plasmids. With our newly transformed strains we infected them with P1 to allow P1 to package the first stage assemblies, creating OCTOphage. With the phage lysate containing OCTOphage, we infected wild-type *E. coli* to deliver our genes. As OCTOphage delivers the genes, wild-type P1 will also lyse the cells. In this second lysate, our enzymes should have been released into the surrounding media. The second lysate was then run on our enzyme assays. To measure plastic degradation we used a weighing assay and for oil emulsification we used a foam stability assay. After we collected measurements from these assays, we compared the data to our positive controls and negative controls. The positive controls were the same enzyme assays with purified protein and for the negative control we ran our enzyme assays with a lysate from wild-type *E. coli*.

### Learn, Redesign, and Success

    While creating our first stage assemblies, we learned about how to properly verify successful results and how to alter the assemblies better for our genes. For each round of Golden Gate Assembly, we transformed our products into *E. coli* and then plated to check for colonies. These colonies would only grow if they were successfully inserted into our vector with the antibiotic resistance and they should be non-fluorescent because we used a RFP drop-out backbone. We also sequenced our products to check for mutations. When we tried to plate our first stage assembly transformed *E. coli*, we didn't get any colonies. When we checked the sequence, we realized that our promoter was mutated and learned that our metabolic burden was most likely too high for our cells. To account for this, we altered our original assembly with a weaker promoter and lower copy number plasmid.

    Once we obtained a working Golden Gate assembly, we needed to determine if cells containing the plasmid would be able to produce enough enzyme to degrade plastic or disperse oil. We did this by conducting SEM and HPLC assays. SEM images yielded significant results. When PET film was incubated with cells expressing our plastic-degrading construct for seven days, the plastic's surface was degraded. This led to a rough, scarred topography as material was broken down.

![](https://static.igem.org/mediawiki/2021/b/b0/T--Austin_UTexas--NegativeInc.png)![](https://static.igem.org/mediawiki/2021/e/e2/T--Austin_UTexas--7Day.png)

The image shown on the left is our negative control, PET which has not been exposed to the plastic-degrading plasmid. The image on the right shows PET degraded by our plastic-degrading plasmid This sample has a significantly rougher surface, with scarring covering its entirety. This shows that our plasmid can be used to effectively degrade PET.

    In addition, in order to create our positive control, we needed to purify our candidate enzymes: PETase, Latherin, Ranaspumin-2 and iLOV-Rsn2. We used IPTG-inducible PET plasmids and 6xHis-tag purification however it was the first time anyone on our team had done the procedure. When we initially tried to express our enzymes and purify them, we didn't see any thick bands when we ran an SDS-PAGE gel. After going back and reading through the parts page for our enzymes we realized that they weren't stable at the temperature that we were incubating them at for induction (37C), so we altered the temperature to 16C which was recommended and successful for another iGEM team. To improve our induction, we also performed induction tests for each enzyme to see which concentration of IPTG would provide the best expression of our proteins. Furthermore, originally we tried lysing our cells using a detergent but we switched to a sonication procedure because we learned that our detergent might not properly isolate the proteins. We used the insoluble fraction of our purification but the detergent may have kept them in the soluble phase instead given the nature of our surfactant proteins. Lastly, we found that our proteins were toxic to the strain of *E. coli* we originally used (*E. coli* BL21) so we switched to SHuffle *E. coli* which would be able to withstand the proteins and also provide the correct reducing environments for our proteins to fold. More on this can be found in the Results page.

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# Team:Austin UTexas/Experiments

# Experiments and Protocols

[Transformation by Electroporation](https://barricklab.org/twiki/bin/view/Lab/ProtocolsElectrocompetentCells)

[Chemical Transformation](https://barricklab.org/twiki/bin/view/Lab/ProtocolsChemCompCellsHub)

[PCR Procedure](https://barricklab.org/twiki/bin/view/Lab/ProtocolsStandardPCR)

[Phage Lysate](https://barricklab.org/twiki/bin/view/Lab/ProtocolPhageLysate)

[Phage Titer](https://barricklab.org/twiki/bin/view/Lab/ProtocolsPhageTiters)

[Golden Gate Protocol](https://barricklab.org/twiki/bin/view/Lab/GoldenGateAssemblyProtocolsMainPage)

[Plastic Degradation Assay w/ HPLC](https://static.igem.org/mediawiki/2021/f/f8/T--Austin_UTexas--HPLC_method.png)

[Bubbling Assay](https://doi.org/10.1016/j.colsurfa.2017.01.049)

[Oil Spreading Assay](https://doi.org/10.1016/S1388-1981%2800%2900124-4)

[Plastic Degradation Assay by Weighing](https://static.igem.org/mediawiki/2021/9/95/T--Austin_UTexas--plastic_weighing_assay.pdf)

[Detergent Lysis](https://www.thermofisher.com/document-connect/document-connect.html?url=https%3A%2F%2Fassets.thermofisher.com%2FTFS-Assets%2FLSG%2Fmanuals%2FMAN0011343_BPER_Bacterial_Protein_Extract_Reag_UG.pdf)

[His-Tag Procedure](https://www.thermofisher.com/document-connect/document-connect.html?url=https%3A%2F%2Fassets.thermofisher.com%2FTFS-Assets%2FLSG%2Fmanuals%2FMAN0011699_HisPur_NiNTA_SpinColumn_UG.pdf&title=VXNlciBHdWlkZTogIEhpc1B1ciBOaS1OVEEgU3BpbiBDb2x1bW5z)

[SDS Page and Coomassie Stain](http://assets.thermofisher.com/TFS-Assets/LSG/manuals/nupage_tech_man.pdf)

[Cassette Dialysis](https://www.thermofisher.com/document-connect/document-connect.html?url=https%3A%2F%2Fassets.thermofisher.com%2FTFS-Assets%2FLSG%2Fmanuals%2FMAN0011668_SlideALyzer_G2_Dialy_Cassette_UG.pdf&title=VXNlciBHdWlkZTogIFNsaWRlLUEtTHl6ZXIgRzIgRGlhbHlzaXMgQ2Fzc2V0dGU=)

[Thrombin Cleavage](https://static.igem.org/mediawiki/2021/9/92/T--Austin_UTexas--thrombin_cleavage_procedure.pdf)

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# Team:Austin UTexas/Results

# Plastic Results

**Plastic Degradation Assays**

We implemented several assays to assess our plasmid’s effectiveness in degrading PET. For these assays, we made measurements using several positive and negative controls in order to compare our system's performance . All assays were tested using samples cultured in either media or buffer containing a PET film, over the course of three to seven days at 30°C or 16°C.

**Negative Control**
First, we created two negative controls for comparison of our experimental results. Our first control was PET film immediately after collection, with no exposure to any kind of incubation or media at all. The second control consisted of PET film placed in cell culture media, which was incubated for three days. No cells nor purified enzyme were included, so no degradation was expected from this sample. PET film typically has a smooth surface on a microscopic level, but degradation of PET by PETase can cause the plastic to develop a rough, scarred surface. Scanning Electron Microscopy (SEM) was conducted on these samples to determine the baseline for PET's texture on a microscopic level, while also determining whether incubation led to any change in texture that could be misinterpreted as degradation of the film. Results for SEM for negative controls were as follows:

![](https://static.igem.org/mediawiki/2021/2/2f/T--Austin_UTexas--NegativeFresh.png)
![](https://static.igem.org/mediawiki/2021/b/b0/T--Austin_UTexas--NegativeInc.png)

**Figure 1: SEM images of negative controls** (A) PET film prior to any exposure. (B) PET film with exposure to cell culture media without PETase.

In these cases, no difference was observed. Shown left is an image of PET film not exposed to any medium nor incubated. Shown right is an image of PET film exposed to LB growth medium for three days. No difference is observed between these samples, showing that our construct is necessary to detect a change in surface topography.

**Positive Control**
As a positive control, we incubated PET film in LB media with purified wildtype PETase. LB media was used to match the conditions used to test our actual phagemid system. The reaction proceeded for three days before measurements were taken. This control was useful for understanding how our experimental samples compare to more concentrated PETase. Similarly, SEM was conducted on this sample and appeared as follows:

![](https://static.igem.org/mediawiki/2021/5/5a/T--Austin_UTexas--Purified.png)

**Figure 2: SEM image of positive control.** Purified WT PETase after incubation for 36h at 30°C at concentration of 200nM in 200uL reaction volume

PET film incubated with purified PETase showed a marked change in surface texture. Holes appear in the plastic’s surface as PETase acts upon it, showing that degradation has occurred. This sample shows that our construct should cause a similar effect if it functions correctly.

High performance liquid chromatography (HPLC) is a common technique used to identify and quantify components in a mixture. In this assay, HPLC was used to identify and quantify the plastic degradation products, TPA and MHET. These monomers are released upon PETase activity. Thus, greater concentrations of PET monomers released would indicate greater degradation.
HPLC measurements were taken for both the purified enzyme, WT PETase, and our second positive control, *I. sakaiensis* at incubation temperatures of 16°C and 30°C. Data was taken at various time points and a linear regression was performed, as indicated by the graph below :

![](https://static.igem.org/mediawiki/2021/0/05/T--Austin_Utexas--HPLC_Final.png)

**Figure 3: HPLC measurements of positive controls**  purified WT PETase in LB media and I. Sakaiensis culture at 30°C and 16°C.

Purified WT PETase performed as expected, with the most degradation over time. Similarly, the other positive control, the *I. sakaiensis* culture also performed as expected. While there were very few PET monomers detected, this is largely due to the fact that*I. Sakaiensis* uses these PET monomers as a carbon source and therefore would not be detected in solution. This does not mean degradation has not occurred. Instead, to confirm PET degradation, SEM images and weight changes were assessed.

**Experimental Samples**
Experimental samples included cells containing our construct. Our construct contained [BBa\_K3601017](http://parts.igem.org/Part%3ABBa_K3601017), a form of PETase and MHETase. This plasmid was expressed by *E. coli* strain C41(DE3) cells incubated in cell culture media for three days.

![](https://static.igem.org/mediawiki/2021/e/e2/T--Austin_UTexas--7Day.png)

**Figure 4:**  SEM image of experimental sample. PET film incubated with c41(DE3) cells expressing our plasmid construct.

Another sample was allowed to incubate for seven days instead. This sample contained our PETase-expressing construct. In this case, degradation is so strong that holes in the plastic’s surface eventually widen to encompass the entire surface.

**Conclusion**

Results from the SEM data validate the activity of our plastic-degrading plasmids compared to the negative controls. This indicates a strong potential for using our P1 system to deliver these plasmids to a variety of bacteria within the bacteriophage's broad host range in order to degrade plastic in the environment. Next steps for the project include using the P1 phage system to deliver these plasmids and assessing activity through HPLC and weight changes. This degradation activity of our proposed system will be measured against the positive controls of the purified enzyme WT PETase and  *I. sakaiensis*  to determine the effectiveness of our system.

# Surfactants Results

**Protein Purification**

In order to ensure the efficacy of our proteins of interest we purified them in high amounts. We did this by over expressing the genes of interest using IPTG-inducible pET plasmids. We began with BL21 DE3 *E. coli* attempting to express Ranaspumin-2, iLOV-Ranaspumin-2, Latherin, and the plastic degrading enzyme PETase, but were only able to express iLOV-Rsn2. We purified the iLOV-Rsn2 using 6xHis-tag and verified this process by checking for iLOV fluorescence and running an SDS-PAGE gel (Figure 1). Figure 1 shows the successful purification of iLOV-Rsn2. Significant amounts of proteins were seen in all 3 elutions slightly larger than 25 kDa, consistent with the expected protein size (iLOV-Rsn2 is 26.7 kDa).

![](https://static.igem.org/mediawiki/2021/9/9b/T--Austin_UTexas--HisTagPAGE.jpg)

**Figure 1: SDS-PAGE gel of His-tag purification of iLOV-Rsn2.** The lanes from left to right are as follows: low molecular weight protein ladder, protein lysate, initial column flowthrough, wash 1, wash 2, wash 3, elution 1, elution 2, and elution 3.

We tried a variety of other cells that contain T7 machinery (to work with the inducible system and promoter) to express our other proteins of interest (PETase, Rsn2, and Latherin) but were still only able to over express iLOV-Rsn2.

**Oil Spreading Assay**

The oil spreading assay directly tests the oil dispersing abilities of a sample. The sample is added to the center of plate containing a thin layer of crude oil covering a much larger volume of water. After a few seconds, an area without oil forms from the point where the sample was added. The area of this "spread" directly correlates to the concentration of surfactant. The Triton X-100 created a large spread while the BSA did not, showing that the assay is a valid method of testing for oil dispersing abilities in a liquid sample. When we tested iLOV-Rsn2, there was a small spread.

![](https://static.igem.org/mediawiki/2021/5/55/T--Austin_UTexas--OilSpreading.png)

**Figure 2: Oil spreading assay.**Top left: 1 mg/mL BSA (a non-surfactant protein), Top right: 0.1% Triton X-100 (a chemical surfactant), and Bottom: 1 mg/mL iLOV-Rsn2 that we managed to purify.

We hypothesize that this reduced dispersion compared to previous data regarding Rsn2 is due to the iLOV tag. Surfactant proteins' conformations are very important due to the unfolding mechanism they use when reducing the surface tension of an interface, so the addition of the iLOV tag to Rsn2 may interfere with its ability to disperse oil.

**Foam Stability/Bubbling Assay**

A more sensitive test for surfactant abilities is the foam stability or bubbling assay. This assay takes advantage of the surfactant ability to decrease the surface tension at an air-water (or solution) interface. This assay involves bubbling large amounts of air through the sample and then determining the stability of the foam created by checking how long it lasts. Because the air-water interface is more miscible than the oil-water interface, this assay is more sensitive to surfactant abilities than the oil spreading assay. We used the same samples as the oil spreading assay but got much better results. The Triton X-100 created a stable foam with small bubbles that popped at a steady but slow rate until the last few layers, which remained for over an hour. The BSA on the other hand did not create a stable foam, rather forming large bubbles that popped quickly. When we tested iLOV-Rsn2, we observed similar foam creation and stability to Triton X-100.

[

](https://static.igem.org/mediawiki/2021/7/75/T--Austin_UTexas--ILOVRsn2andBSA.mp4)

**Figure 3: Timelapse of iLOV-Rsn2 (left) and BSA (right) over 10 minutes**

[

](https://static.igem.org/mediawiki/2021/4/41/T--Austin_UTexas--ILOVRsn2andTriton.mp4)

**Figure 4: Timelapse of iLOV-Rsn2 (left) and Triton X-100 (right) over 20 minutes**

**Conclusion**

Although we were unable to correctly assemble the phagemid for the surfactant genes Rsn2 or iLOV-Rsn2, we were able to verify that iLOV-Rsn2 maintains some of the surfactant ability of untagged Rsn2. This was shown by both the oil spreading assay and the foam stability/bubbling assay. The oil spreading assay with iLOV-Rsn2 sample showed a nontrivial spread, indicating that iLOV-Rsn2 does display surfactant abilities despite the addition of the iLOV tag potentially affecting its conformation and unfolding mechanism. The foam stability assay further enforced this when the iLOV-Rsn2 sample displayed bubble formation and foam stability similar to the known chemical surfactant Triton X-100. This indicates a strong potential that upon replication with untagged Rsn2, the dispersion may be strong enough to effectively disperse oil slicks.