Team:IIT Chicago/Design

iGEM IIT Chicago

Components

Green Ocean has developed multiple designs in the creation of our product. You can click on any of the following components below to learn more about them.

Primer Design

Take a look at the primer design to understand the initial foundation of DNA replication process of our superPETase gene.

Super PETase Design

If you are interested in the superPETase gene, please click here to understand the development of it

Assay Design

Understand more on how we designed our test to verify that our enzyme can break down PET (polyethylene terephthalate) plastics in the ocean

pAM and PET Fragment Primers

Design Methods

The two plasmids being used for developing this specific cyanobacterium are pAM4788 as the backbone vector and pCS-PET_VB190412 for the superPETase gene. For convenience’s sake, they shall be referred to in this document as pAM and PET, respectively. In order to design these primers, two steps must be taken. The first step is to identify the regions of each plasmid necessary for PCR and Gibson cloning. The second step is to isolate the specific fragments of each plasmid that will be used in the PCR and cloning protocols.

Region Identification

In the PET plasmid, the region to be isolated is that of the designed superPETase gene (shown in yellow in Figure 1).

Figure 1. Graphic map of the pCS-PET_VB190412 plasmid identifying featured regions.

In the pAM plasmid, the region to be removed is the GFP gene (shown in green in Figure 2), leaving the rest of the plasmid as the backbone vector. The superPETase gene (yellow) is inserted where the GFP gene (green) was removed.

Figure 2. Graphic map of the pAM4788 plasmid identifying featured regions.

PCR Primer Isolation

The basic requirements for a PCR primer sequence is that n ≥ 18 and that Tm ≥ 55°C, where n is the number of base pairs and Tm is the melting temperature. A forward and reverse primer must be made for each fragment that is desired to be replicated. For the superPETase gene, the primers would be composed of the sequences of base pairs at the beginning and ending of the gene. For the pAM plasmid backbone, the primers would be composed of the sequences of base pairs just before and just after the GFP gene. Running PCR amplification for the pAM backbone consist of >7k base pairs. This is too large to be efficient. To compensate for this, we divided the pAM backbone into a variety of separate fragments and created primers.

Gibson Primer Design

For Gibson primers, fragments of the two different plasmids being joined must be combined into one primer that will overlap regions of the backbone and product vectors. The basic requirements here are that n ≥ 20 and Tm ≥ 50°C. For identification purposes, these primers will be labeled in an A-B_d fashion, where A is the whole fragment from one plasmid, B is the plasmid to which incomplete fragments of A are attached to the other whole fragment, and d is the direction (Figure 3).

Figure 3. Schematic description of Gibson primer structure and naming convention.

Internal PETase Primers

Strategy

We do initial screening with the sequencing primers that flack the gene insertion site in the vector backbone. Every plasmid backbone has a _f and_r sequencing primer which is used to sequencing interred genes, and can also be used to PCR the interest up. When screening by PCR we look for a specific size (size of insert plus flanking sequence). However this does not provide specific amplification due to the gene, and might be misleading if there is an insert of similar size.

To confirm OUR gene is inserted, we want a specific primer, which will ONLY amplify our gene of interest. This will be a lot more diagnostic. We can do this by making primers specific to the gene, and using one of these as the _f or _r primers in conjunction with the sequencing _r an d _f primers.

Primer Summary

Name Template Forward Primer Reverse Primer Size (bp)
PET pCS_PET pAM_PET_f PET_pAM_r 1034
Frag A pAM pAM_F2 pAM_35k_r 3521
Frag B pAM pAM_35k_f pAM_r 3554
Table 1. Shows the 3 fragments used to make pAM_PET
pAM_f2 AAGGGTGGGCGCGCCGACCCAGCTTT
pAM_r GGT GAA GGG CTC CTT CTT AAA G
PET-pAM_r AAT AGG TCG TTG TTT GCC ATG GTG AAG GGC TCC TTC TTA A
pAM-PET_f TTA AGA AGG AGC CCT TCA CCA TGG CAA ACA ACG ACC TAT T
pAM35k_f CGA ATT GAT CGG CAA GCC AG
pAM35k_r CTG GCT TGC CGA TCA ATT CG
Table 2. Specific primer sequences of final primers used for fragmentation and screening.

More Information

Design Purpose

The purpose of the superPETase vector is to possess the PETase gene and be able to properly function and be expressed by cyanobacteria, our host.

Design Methods

Two genes were chosen from previous research and were modified to be expressed in the desired host.

  1. SuperPETase sequence
  2. TorA sequence.

Super PETase

PETase gene sequence was obtained from a research paper (1). The sequence obtained is as follows:

In the original paper it was mentioned that the gene was mutated to enhance PETase activity, but the gene sequence did not show this change. We therefore had to make the sequence change. In order to enhance activity I had to be changed to F. We had to look at the primers and look for the I we need. The primers are as follows:

The only difference between these primers are a V and F and by looking at the codons we can identify where V and F are located and identify the reading frame.

We then translated these codons to identify the flanking amino acids

We then look at the original sequence and find this: CENDSIAPVNS

We have found our I and now we can change the I to F:

We have now made the IsPETase gene to SuperPETase.

TorA

In order for S. elongatus to be able to express and deliver the SuperPETase gene we need to use an excretion pathway from S. elongatus. Studies have shown that tat pathway in S. elongatus is used in cyanobacteria (2). In order to use this TorA sequence was used. TorA is a signal peptide that would aide on the excretion of SuperPETase.

The TorA sequence is: MANNDLFQASRRRFLAQLGGLTVAGMLGPSLLTPRRATA

Which translate to the following codons:

Both genes were then added to a vector that was created by VectorBuilder. The gene sequence is as follows.

In orange you will find the TorA sequence. Followed by the substitution of the start codon to ser highlighted in yellow. The PETase gene is found in violet and in the gene you will find the mutation "TTC", and a myc tag in green color

Citations

  • 1. Ma, Y., Yao, M., Li, B., Ding, M., He, B., Chen, S., Zhou, X., and Yuan, Y. (2018) Enhanced Poly(ethylene terephthalate) Hydrolase Activity by Protein Engineering. Engineering. 4, 888–893
  • 2. Spence, E., Sarcina, M., Ray, N., Møller, S. G., Mullineaux, C. W., and Robinson, C. (2003) Membrane-specific targeting of green fluorescent protein by the Tat pathway in the cyanobacterium Synechocystis PCC6803. Molecular Microbiology. 48, 1481–1489

The purpose of the assay team is to design and perform tests verifying that our enzyme can break down PET (polyethylene terephthalate) plastics in the ocean.

Nanoparticles of PET were designed as test subjects, obtained in two ways. Firstly, 2-liter soda bottles were filed and shaved down, providing a “real-world” example of what’s actually in the ocean. Some commercial bottles, however, contain plastics other than PET, and other modifiers, so clean, commercially PET was purchased as small pellets.

To produce PET nanoparticles a dissolution / precipitation/ settling protocol was developed based on prior work. This entailed dissolving PET in in 90% TFA 10% water. Contrary to prior studies we found we did not need to mechanically disrupt macroscopic PET to do this. This was then slowly diluted over >12 hours by dropping into an equal volume of 10% TFA in water. These were then and washed, sonicated and washed again. A fill protocol is provided in the protocol tabs or a quick overview in the image below.

For labeled nanoparticles Fluorescein was added to the PET dissolved in 90% TFA. It is then trapped in the PET particles as they precipitate when it is diluted into water, creating essentially a solid solution. As the PET is degraded, the fluorescein is released into the solvent. Degradation can then be monitored by release of gluorescncesin ot the aquesous pahse, or by other spectroscopic changes such as the polarization of the fluorescein which is very different in water vs in solid solution in PET due to exterment large viscosity differences.

This result dina heterogeneous preparation for PET on the 10nm to 1mm scale. Larger particles were removed by 90% Fluorescein, a common fluorophore, was then added to the nanoparticles. The fluorescein was dissolved.

Illinois Institute of Technology

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