Team:Linkoping Sweden/Design

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Back to the top Our design CBD Linker Pink-White Choice of agents

Project Design

Background

When a person falls victim to a wound, the body’s immune defense is activated to prevent and fight off invading microorganisms. However, in burn wounds the body’s immune defenses are altered due to reduced oxygen levels in the wound, lowered Immunoglobulin G (IgG) levels and other components in the complement system needed for a fully functioning immune defense. The immune system is also affected due to a loss of tissue which, both in relation to tissue layers and area, can prevent the immune cells to migrate to the affected area and infecting bacteria [1]. Managing these types of wounds has always been a challenge due to the high risk of infections caused by various microorganisms such as bacteria.

Today we treat burn wounds with the aid of high antibiotic doses in order to eradicate bacteria. The problem with this treatment is that a lot of bacteria can develop antibiotic resistance and some bacteria are natively immune to antibiotics.

Our design

Figure 1: Mechanism of action. The carbohydrate binding domain (CBD) is bound to a polysaccharide material (e.g. cellulose). The CBD has below in a linker (GS-linker) which ends in a thrombin cleavage site. On the end of the thrombin site is a antimicrobial agent. When the fusion protein is exposed to thrombin the linker will be cleaved, releasing the agent.

In order to fight off these antibiotic resistant microorganisms we will use antimicrobial agents such as different antimicrobial peptides (AMP) and enzymes. By combining these agents with a carbohydrate binding domain (CBD) via a flexible linker, we created a fusion protein with the property of binding to a polysaccharide bandage, such as cellulose, which will be placed on the wound and kill off the bacteria and make it sterile.

To increase the activity of our AMPs we also inserted a thrombin cleavage site in the linker. When the bandage is exposed to thrombin in the wound, the bandage will release the AMPs into the wound which will increase the reach and activity in the wound and sterilize it as shown in Figure 1.

Expression system

The construct has a strong expression with a T7-RNA-polymerase promotor (BBa_I719005) as well as a 5'-UTR (BBa_K1758100) region which has been shown to further increase expression in E. coli (BBa_K1758106).

Carbohydrate binding domain

The CBD from Clostridium thermocellum cellulose scaffolding protein CipA was chosen due to its strong, but still reversible binding. It is also well documented by previous iGEM teams: Imperial 2014, Edinburgh 2015 and Ecuador 2018. The CBD binding to a polysaccharide material is unaffected in many different solutions, e.g. salt containing- and ethanol solution, while exposure to water will release the CBD as wished.

The CBD is used mainly for two reasons:
1. Enabling easy and quick purification
2. Inactivation of the antimicrobial agent

For the first reason, the fusion protein can directly bind to polysaccharide containing material after lysating the bacteria, therefore the polysaccharide material can directly be incubated to retrieve the protein. This eases the purification step by quickly retrieving the protein afterwards, the polysaccharide containing material can be washed with solutions such as ethanol to release nonspecifically bound proteins. The CBD purification is similar to any other affinity purification. The second reason is that the CBD will inactivate the antimicrobial agents, primarily antimicrobial peptides (AMP). These AMPs cannot be expressed as they are, they need to be inactivated to inhibit them from harming the expressing bacteria. The CBD is hydrophilic while the peptides are hydrophobic, therefore, the CBD will act as an anchor to the AMP.

The CBD was also chosen due to its wide ability to bind almost any polysaccharide containing material. We wanted to create an antimicrobial bandage and a newly used material is cellulose. Cellulose does not create an immune response.

Figure 2: Proposed wash steps of the bandage. Cellulose, or any other polysaccharide containing material can be incubated in bacterial lysate from an bacteria which has expressed our construct. The carbohydrate binding domain (CBD) will associate to the cellulose and the fusion protein can therefore be purified from the lysate. Afterwards, to release non-specifically bound proteins, the cellulose is washed 3 times in 70% ethanol.

Linker

The CBD C-terminal fusion contains a flexible GS-linker (-GGGGSGGGGS-). A thrombin cleavage site (-LVPRGS-) was added to the end of the linker and its breakage will leave a glycine and serine attached to the N-terminal of the released fusion protein (agents in this case). The cleavage will result in an activation of the antimicrobial peptide and increase the reach of the enzymes. And according to our model, the glycine and serine residue will not affect the antimicrobial activity of our peptides.

In our use, a patient’s own blood which contains thrombin will cleave the linker and activate the agent allowing it to access deeper lying bacteria. The thrombin site also contains a BamHI site. This is to be able to allow an easy change of the antimicrobial agent or any other protein by having a BamHI site on the 5’-end and biobrick suffix in the 3’-end. This allows the user to only needing to order a smaller fragment than needing to order the whole construct containing the CBD and linker. The internal BamHI cleavage site codes for glycine and serine, fitting it to the end of the thrombin site. It is also a cost-effective enzyme and is unaffected by methylated DNA.

Figure 3: Construct overview. The construct is composed of a carbohydrate binding domain (CBD) with a linker containing a thrombin and BamHI site on the C-terminal (3'-end). The BamHI-site on the end of the linker allows exchange of fusion protein. With a pSB1C3 backbone the enzymes needed are BamHI and PstI or SpeI. A more detailed example of how to switch fusion protein can be observed below in Figure 4.

Pink-White Screening

We designed a construct containing CBD-pCons-AsPink, leading to pCons-AsPink being a dropout. This enables colour-screening (pink-white) for positive colonies. Using BamHI and PstI or SpeI on the pSB1C3-CBD-pCons-AsPink vector, the pCons-AsPink sequence will be dropped out of the construct, allowing the compatible sequence of choice to ligate to the empty CBD-linker vector. This will result in a vector containing the newly ligated insert (such as GFP) fused in frame with a linker to the CBD. The fusion protein can later be cleaved with thrombin to yield two separate proteins or to purify the protein via the CBD (such as done with His-tags). The C-terminal fusion will have one glycine and one serine added to the N-terminal of the protein.

One can design any gene to be fused with the CBDcipA as long as it contains a BamHI recognition sequence in the 5'-end. The biobrick suffix can be used in the 3'-end. Then one can cleave the vector and insert with BamHI and PstI (or Spel), with no need for gel purification. Mix and add the new insert and transform a bacterial host, the white colonies the next day has the correct fusion protein. The pink colonies has been re-ligated and contains the base CBD-pCons-AsPink vector.

Figure 4: Walkthrough of Pink-White screening. The original plasmid contains two promotors: T7 and pCons. pCons is constitutive, meaning that AsPink alone will always be expressed in the bacteria, leading to pink colonies. When using BamHI together with SpeI or PstI, the pCons-AsPink construct will drop out. Afterwards, a construct that has previously been cleaved with the same restriction enzymes can be ligated to the vector, creating a fusion protein (shown here as a antimicrobial agent (purple), as illustrated in Figure 1. When attempting the exchange from pCons-AsPink to fusion protein of choice, the colonies containing the original construct with pCons-AsPink will be pink while the successfully ligated vectors will turn white. This can be compared to the more known blue-white screening using lacZ.

Explaining Our Choice of Antimicrobial Peptides

The peptides chosen come from a broad spectrum of organisms. LL-37 is a human AMP that is used by the innate immune system. Magainin 2 is expressed by the African clawed frog and Pln1 is expressed by the bacteria Lactobacillus plantarum. We aimed to have a broad spectrum of peptides due to the fact that they might be more or less toxic when expressed in bacteria and might be inactivated in different degrees by the CBD. The AMPs were also chosen due to the fact that they might have different efficiency against bacteria mainly because they have different mechanisms when acting on the bacterial membrane.

Explaining Our Choice of Antimicrobial Enzymes

We also choose to have antimicrobial enzymes, the ones used by us are bacteriophage lysins. We choose enzymes due to their possibility of higher activity and specificity. After feedback from meetings with Uppsala (Akademiska) and Linköping University hospital’s burn care centers we realized that the ESKAPE group of bacteria is problematic therefore we choose to have two enzymes mainly targeting two of the ESKAPE bacteria.

CHAP (C1-162) mainly target S. aureus and PlyF307 targets A. Baumanii. CHAP (C1-162) is an enzyme derived from Staphylococcus phage lysin. The LysK consists of a C-terminal cell wall binding domain and an N-terminal catalytic domain which attacks the peptidoglycan in the bacterial cell wall. In our project we chose to discard the C-terminal binding domain and only choose the CHAP domain (C1-162) of LysK to increase the antimicrobial activity against gram positive bacteria [2].

PlyF307 is composed of an amidase domain spanning amino acids 1-103, it also has an AMP afterwards (called P307): 104-147 which assist the protein in finding and bringing the amidase domain to the cell wall of the bacteria. A previous study observed that by mutation of the last eight amino acids of the AMP into a Hepatitis B virus peptide, the activity could be increased. We chose to add this hepatitis B virus mutation to the whole PlyF307. This has only been studied for the P307 peptide without the amidase domain before [3].

References

1. Eming SA, Martin P, Tomic-Canic M. Wound repair and regeneration: mechanisms, signaling, and translation. Sci Transl Med. 2014 Dec 3;6(265):265sr6.
2. Horgan M, O’Flynn G, Garry J, Cooney J, Coffey A, Fitzgerald GF, et al. Phage Lysin LysK Can Be Truncated to Its CHAP Domain and Retain Lytic Activity against Live Antibiotic-Resistant Staphylococci. Appl Environ Microbiol. 2009 Feb 1;75(3):872–4.
3. Thandar M, Lood R, Winer BY, Deutsch DR, Euler CW, Fischetti VA. Novel engineered peptides of a phage lysin as effective antimicrobials against multidrug-resistant Acinetobacter baumannii. Antimicrob Agents Chemother. 2016;

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