Design
Diagram of complete system design implemented in a generalized cell.
Arabinose Induction:
Figure 1.
Diagram of Arabinose (Ara) Operon [1]
Some of our constructed parts use the pBAD promoter which is taken from the arabinose operon native to E. coli. Arabinose is transported into E. coli cells by the integral membrane proteins AraE and AraFGH. Transport is most optimal in the absence of the preferred sugar lactose, which causes catabolite repression [2].
Figure 2.
Diagram of Arabinose Transport in E. coli [3]
Intracellular arabinose then causes a conformational change in the transcriptional activator AraC. Activated AraC - Arabinose then binds to the pBAD promoter region and activates transcription of the downstream genes AraB, AraA, and AraD which are responsible for the breakdown of arabinose into a chemical form the cell can use metabolically. The pBAD promoter is tightly regulated by AraC-Arabinose [4] and is commonly used as an inducible promoter for experiments.
Figure 3.
Diagram of AraC-Arabinose allosteric regulation of the pBAD promoter [5]
Sensing
Quorum sensing is a method of cell communication by which individual cells produce and secrete unique molecules that act as intercellular chemical signals. These molecules bind to the receiving cell using various mechanisms, such as surface receptor proteins embedded in the cellular membrane, and can then modulate intracellular activity within the individual bacterium. A variety of intracellular responses creating changes in gene expression can be produced depending on the type of chemical signal received.
Figure 4.
Diagram of Agr Quorum Sensing System in S. Aureus [6]
Staphylococcus Aureus uses a set of genes called the Accessory Gene Regulator (Agr) System in their quorum sensing communication. AgrB and AgrD are responsible for producing the chemical messenger molecule of the species called autoinducing peptide (AIP). AIP-1 functions as an activator of the Agr system. AgrC is an integral membrane protein consisting of 6 alpha helices, an intracellular histidine kinase domain, and an extracellular receptor domain that detects AIP from the environment [7]. There are four types of AIP produced by S. Aureus and four respective versions of the AgrC gene which encode different derivations of the AgrC protein with drastically different sequences in the N-terminal transmembrane domain [8].
Figure 5.
Alternative Structures of all Four AIP Derivatives [9]
When AIP-1 binds to AgrC it results in a conformational change on the cytoplasmic side of the protein and catalyzes the exchange of a phosphate group from AgrC to AgrA. Upon phosphorylation, AgrA dimerizes to its active form and functions as a transcriptional activator. AgrA interacts with the intergenic region between the P2 and P3 promoters found in S. Aureus and binds a DNA motif at two different sites upstream of the P2 promoter [10,11]. Once bound, the AgrA dimer creates a conformational change in the DNA that allows RNA polymerase to bind to either promoter [12]. The promoter chosen is based on additional signalling conditions. The genes downstream of P2 are responsible for the Agr proteins, while the genes downstream of P3 are responsible for the harmful toxins of S. Aureus and are only produced when needed.
Sar is another operon activated in response to environmental signals and produces the proteins SarA and SarR [13]. These two proteins act in conjunction with AgrA to either upregulate (activate) or downregulate (repress) the level of transcription experienced at either promoter site. AgrA is required for activation while the presence of either SarA or SarR is optional and only further modulate transcription. SarA, the activator, causes an additional bend in the conformation of the DNA in the intergenic region. SarR has a higher binding affinity than SarA, and can displace it in order to linearize the DNA into a non optimal form [12].
Figure 6.
Proposed Activation Mechanism of P2 Promoter by Transcription Factors: [12]
To implement the Agr quorum sensing system into E. coli, only the AgrC and AgrA genes need to be introduced to the E. coli and any desired genes may be placed downstream of the P2 promoter. In this way, E. coli will be able to detect and respond to an AIP-1 signal produced by S. Aureus. AgrB and AgrD need not be included because then the E. coli would produce their own AIP-1 which could cause interference. Additionally, the P2 promoter is used rather than the P3 promoter because AgrA has a higher binding affinity with the P2 promoter when phosphorylated [10] and because of P3’s association with virulence genes.
Agr quorum sensing was first attempted by iGEM Cambridge 2007 who placed AgrC and AgrA (BBa_I746100) downstream of an inducible pBAD promoter (BBa_206000) which is controlled by arabinose. iGEM Tu Delft 2013 then improved the system by adding a GFP reporter (BBa_E0040) downstream of a P2 promoter (BBa_I746104) in order to detect if the sensing system was successfully being induced by the addition of AIP-1. Tu Delft’s composite plasmid construct is called BBa_K1022100.
Figure 7. TuDelf Agr System Plasmid
Source: Tu Delft iGEM 2013
Tu Delft produced results with a marginal level of statistical significance when testing the level of GFP fluorescence intensity using flow cytometry. They compared BBa_K1022100 both induced and uninduced by AIP-1 to a negative control with no GFP gene and a positive control that expresses GFP Constitutively. They found that their system did respond to induction by an AIP-1 signal, but the level of fluorescence intensity was much lower than that of constitutively expressed GFP. This suggests the level of transcription produced by the P2 promoter is much lower than the constitutive promoters currently available. This negatively impacts the ability of the sensing system to successfully and effectively produce a regulated response to S. aureus.
We aim to improve upon the work previously done by these two teams. SarA has been shown by previous research to further activate the transcription at the P2 promoter in conjunction with AgrA. By adding this gene to the system, we should observe a level of transcription, measured by GFP fluorescence intensity, higher than that of AgrA alone and closer to that of cells with GFP expressed constitutively.
Furthermore, during our research we also discovered that the original sequences for AgrA, AgrC, and the P2 promoter listed by iGEM Cambridge 2007 are all incorrect. Cambridge is cited as having taken their sequences from Staphylococcus aureus strain NCTC 8325, a commonly used strain for genetic manipulation. However, when looking at the entirety of the sequenced genome, the protein sequences for both agrC and agrA, and the region denoted for the P2 promoter, are all shorter than almost every other strain of S. aureus compared using NCBI Blast. This would indicate that the strain NCTC 8325’s original annotation found from its 1988 paper is incorrect compared to modern results. Cambridge offered no documented explanation for these changes. The shortened sequences could have profound negative implications on the proper functioning of the proteins and the level of expression of the P2 promoter. This may explain why teams in years past have produced mixed results when using this Agr sensing system.
AgrC - Strain NCTC 8325/Cambridge:
MILMFTIPAI ISGIKYSKLD YFFIIVISTL SLFLFKMFDS ASLIILTSFI IIMYFVKIKW YSILLIMTSQ IILYCANYMY IVIYAYITKI SDSIFVIFPS FFVVYVTISI LFSYIINRVL KKISTPYLIL NKGFLIVIST ILLLTFSLFF FYSQINSDEA KVIRQYSFIF IGITIFLSIL TFVISQFLLK EMKYKRNQEE IETYYEYTLK IEAINNEMRK FRHDYVNILT TLSEYIREDD MPGLRDYFNK NIVPMKDNLQ MNAIKLNGIE NLKVREIKGL ITAKILRAQE MNIPISIEIP DEVSSINLNM IDLSRSIGII LDNAIEASTE IDDPIIRVAF IESENSVTFI VMNKCADDIP RIHELFQESF STKGEGRGLG LSTLKEIADN ADNVLLDTII ENGFFIQKVE IINN
= 414 amino acids
Uniprot and NCBI Sequence:
The highlighted region shows that the first 16 amino acids are missing from the finalized protein in 2007 Cambridge’s/2013 Tu Delft’s construct.
Faulty AgrC Implications:
Figure 8.
Acetabularia acetabulum rhodopsin AR2 protein analogous to AgrC
AR2 Modeled in UCSD Chimera (PDB: 3AM6)
The first 16 amino acids have been highlighted in a protein closely related to AgrC since the intermembrane portion has never been successfully imaged by X-ray crystallography at this point in time. Cambridge AgrC would be missing an extracellular limb and ½ of an intermembrane alpha helix. The limb could be important for the binding of AIP-1 from the environment, and the helix located on the lateral side of the molecule would interact directly with the cell membrane and could cause problems with proper placement and incorporation if missing.
AgrA - Strain NCTC 8352/Cambridge
MEIALATDNP YEVLEQAKNM NDIGCYFLDI QLSTDINGIK LGSEIRKHDP VGNIIFVTSH SELTYLTFVY KVAAMDFIFK DDPAELRTRI IDCLETAHTR LQLLSKDNSV ETIELKRGSN SVYVQYDDIM FFESSTKSHR LIAHLDNRQI EFYGNLKELS QLDDRFFRCH NSFVVNRHNI ESIDSKERIV YFKNKEHCYA SVRNVKKI
= 208 amino acids
Uniprot and NCBI sequence:
The highlighted region shows that the first 30 amino acids are missing from the finalized protein in 2007 Cambridge’s/2013 Tu Delft’s construct.
Faulty AgrA Implications:
AgrA modeled in Chimera (PDB: 4XQJ)
The first 30 amino acids are not involved in the interaction of agrA and the DNA of the P2 promoter. However, they could be important for the portion of the protein interacting with AgrC. This region has not yet been imaged by X-ray crystallography.
P2 - Strain NCTC 8325/Cambridge:
ATTAAATACA AATTACATTT AACAGTTAAG TATTTATTTC CTACAGTTAG GCAATATAAT GATAAAAGAT TGTACTAAAT CGTATAATGA CAGTGA
= 96 base pairs
The intergenic sequence between the P2 and P3 promoters is approximately 186 base pairs [12].
Intergenic Region of S. aureus P2 and P3 Promoters [12]
The gene sequence above presents P3 oriented in the 5’ to 3’ direction. The viewing perspective must be changed by taking the reverse complement of the gene sequence to put P2 in the 5’ to 3’ direction. This results in the following sequence:
The highlighted region shows that 90 base pairs are missing from the intergenic region of the promoter in the 2007 Cambridge/2013 TU Delft constructs.
Faulty P2 Implications:
Intergenic Region AgrA binding motifs [11]
Intergenic Region of P2 promoter with Transcription Factors [Adapted from 12]
Although the P2 promoter sequence by Cambridge has the RNA polymerase binding site (-35 to -10) intact, the intergenic sequence is important for transcriptional regulation by the two transcriptional activators AgrA and SarA. The sequence is missing the second SarA binding site and the 3rd and 4th AgrA binding sites (upper half of the loop in the top right picture). This could affect the proper expression of any genes placed downstream of a P2 promoter.
Our Sensing Design:
Plasmid map of Tu Delft’s original Agr Sensing System (BBa_K1022100) reconstructed
Corrected Agr Sensing System (BBa_K3191101) with corrected AgrC (BBa_K3191110), AgrA (BBa_K3191111), and P2 (BBa_K3191400) sequences
Improved Agr Sensing System (BBa_K3191102) with SarA (BBa_K3191100) added to the corrected sequence construct (BBa_K3191101).
We will test these plasmid constructs by measuring the overall fluorescence intensity of the GFP produced by these cells using a plate reader. We will compare Tu-Delft’s Original Agr Sensing System (BBa_K1022100) versus our Corrected Agr Sensing System (BBa_K3191111) and our SarA - Improved Sensing System (BBa_K3191102) using four conditions: No induction, arabinose induction only, AIP induction only, and induction by both chemicals. These constructs will also be compared to data collected from a positive and negative control.
The positive control for sensing: BBa_K608008 - taken from Plate 1, Well 5I in the 2019 iGEM Distribution Kit, uses the same pSB1C3 chloramphenicol vector as most other iGEM parts and contains GFP expressed by a constitutive promoter of the BBa_J23119 family and is intended to emulate a sensing system working at maximum capacity. It will be independently characterized prior to sensing by comparing it to BBa_J23119 - taken from Plate 2, Well 18P in the 2019 iGEM Distribution Kit, contains only the original constitutive promoter by itself in pSB1C3.
The negative control for sensing: BBa_K206000 - taken from Plate 3, Well 13A in the 2019 iGEM Distribution Kit, contains only the pBAD promoter by itself in a pSB1C3 plasmid to emulated a sensing system that cannot possibly be induced. It will be independently characterized prior to sensing by comparing it to BBa_K584000 - taken from Plate 1, Well 7G in the 2019 iGEM Distribution Kit, which contains the pBAD promoter followed by a GFP reporter in a pSB1C3 plasmid.
Motility
E. coli are naturally motile bacteria. Motility can also be referred to as chemotaxis. Chemotaxis is the movement of a cell towards or away from a chemical stimulus based on concentration, either from high to low or from low to high [14]. The family of genes called “Che” are native to E. coli and are located in their chromosomal DNA. Normally, chemotaxis is influenced by the post-translation modulation of the CheY and CheZ proteins. When phosphorylated, CheZ is bound to the motor proteins of the bacterial flagella causing a clockwise rotation that results in the cell “tumbling”. When CheY is dephosphorylated by CheZ, it detaches from the motor proteins allowing the flagella to rotate counterclockwise creating a “running” or swimming” motion [15]. CheZ can be activated by a variety of environmental or intracellular signaling pathways that direct the cell to follow a certain chemical gradient, such as one leading to a source of food like glucose [15].
Figure 12.
Diagram of Che Proteins’ Influence on Flagellar Motility [16]
To demonstrate the use of directed chemotaxis, we elected to use part BBa_K1412614 created by iGEM XMU-China 2014 which features a Copy of CheZ downstream of a pBAD promoter. We have placed their composite CheZ part in a second plasmid vector. This plasmid, pSB4A5, has a low copy number, a different ori site that is compatible with pSB1C3, and ampicillin resistance for selection. A mutant variation of strain K-12 E .coli with its chromosomal CheZ deleted will be used. It has been provided by Dr. Parkinson at the University of Utah, and is the same mutant used in “Reprogramming Microbes to Be Pathogen-Seeking Killers” by Hwang, et al. [15] which inspired our project. By this method the E. coli will be non-motile except when CheZ is transcribed in response to successful activation of the pBAD promoter by AraC-Arabinose. This would allow our E. coli to follow an arabinose gradient streaked on a plate.
XMU China's (2014) plasmid map of a pBAD promoter and CheZ (BBa_K1412614)
Chemotaxis can also be inhibited by the overexpression of CheZ. If the CheZ proteins were to accumulate and linger within the cell, it would cause the cell to swim excessively even when the signal input is no longer present [15]. In order to combat this issue, we have attached a degron to the C-terminus of the CheZ protein. A degron is a sequence of amino acids that marks a protein for degradation [15]. Our degron of choice is called YBAQ, the same as used by Hwang., et al. [15]. The degradation only occurs after a delayed response. This will create a staggered effect of swimming and stopping that should allow the E. coli to continuously reevaluate their situation and the direction of the signal source. This will result in the E. coli following the chemical trail more accurately. Our YBAQ part (BBa_K3191200), includes a stop codon that the existing part (BBa_K2819011) lacks.
Plasmid map of CheZ with YBAQ Degron (BBa_K3191201)
In our finalized system, AIP will provide the chemical gradient necessary to move E. coli in the direction of S. aureus. The pBAD promoter is swapped in favor of the P2 promoter. In this way, CheZ is only transcribed in response to successful activation of the P2 promoter by AIP in the presence of our sensing plasmid. This would allow our E. coli to directly follow an AIP gradient straight back to a source of S. Aureus for extermination.
Plasmid map of BBa_K3191201 with P2 promoter
Killing
Bacteriocins are a class of small peptides with antimicrobial activity that can inhibit or kill other bacteria [17]. Bacteriocins function by damaging the membranes of cells; in comparison, many conventional antibiotics function as enzyme inhibitors [17]. Because of this alternate mode of action, bacteriocins are typically active against antibiotic-sensitive and antibiotic-resistant bacteria [17]. We have designed our system to produce a recently discovered bacteriocin termed garvicin KS (GarKS). GarKS is a three-unit peptide isolated from Lactococcus garvieae, a species found in raw bovine milk [18]. GarKS is encoded by a series of three structural genes, gakA (BBa_K3191310), gakB (BBa_K3191311), and gakC (BBa_K3191312), in an operon-like structure.
Structure of Garvicin KS gene cluster [17]
GarKS has a broad inhibitory spectrum against Gram-positive bacteria, including species like Acinetobacter baumannii, Listeria monocytogenes, and numerous strains of Staphylococcus aureus [19]. GarKS is capable of acting synergistically with other bacteriocins, improving the speed and effectiveness with which it acts against Gram-positive bacteria [19]. Its broad spectrum of inhibition, effectiveness against Gram-positive species, and its synergistic effects make it a viable option for antimicrobial use.
Our initial design places the three GarKS structural genes downstream of a pBAD promoter in an E. coli chassis (BBa_K3191301). This is to encourage GarKS production in the presence of L-arabinose. While our E. coli chassis is a Gram-negative bacteria, the native source of GarKS, L. garvieae, is Gram-positive [17]. This may create issues when attempting to transport the translated GarKS subunits outside the cell. To combat this problem, we have identified a secretion peptide to attach to each of the subunits. Our team has selected a novel signal peptide designed for secretion of S. aureus Alpha toxin H35L [20]. This sequence, designated NSP4 by its designers, tags peptides for the SEC-dependent secretion pathway [20]. The NSP4 sequence (BBa_K3191320) was attached to the N-terminus of each GarKS structural component to create a GarKS + NSP4 composite part (BBa_K3191301).
Plasmid map of BBa_K3191301 contains the three GarKS genes downstream of a pBAD promoter
Plasmid map of BBa_K3191302 with a NSP4 secretion peptide attached to GarKS. The system is under the control of a pBAD promoter
Plasmid map of our Garvicin system with a NSP4 secretion peptide attached to GarKS. The system is under the control by a P2 promoter, allowing the agr system to control GarKS production
Citation
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