Team:Austin UTexas/Engineering
Engineering Success
Summary of Design Cycle
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, pET28-Rsn2, pET28-iLOV-Rsn2) 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
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
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.
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.