• New parts added to the Registry of Standard Biological Parts

Since our project Apt4UTI focuses on developing a novel aptamer-based diagnostic kit for detecting UTI-causing pathogens, we have created a prototype through the designing of our Biobricks parts that will be useful for teams to test their aptamers on E.coli or other uropathogenic bacterial strains that will express a particular protein as either a virulence factor or an adhesive protein. We have also designed several assays by which we can test our parts:

Basic Parts

• FimH1 (BBa_K4449000)

The DNA part contains the coding sequence of the E.coli UTI89 FimH1 signal peptide and lectin domain⁵ which forms a subunit of type 1 fimbriae, located at the tip of the organelle as an integral part of short fimbriae. These structural organelles help the Uropathogens to mediate specific adhesion to alpha -D-mannoside receptors of the host and represent hair-like structures on the surface of E.coli.¹ Therefore, FimH 1 acts as a receptor-recognition element and hence as an adhesin that is responsible for the pathogenesis in urinary tract infection.¹ For further information related to this part please visit our parts page FimH1 (BBa_K4449000)

• FimH2 (BBa_K4449001)

The DNA part contains the coding sequence of the E.coli UTI89 FimH2 signal peptide and lectin domain⁵ which forms a subunit of type 1 fimbriae, located at the tip of the organelle as an integral part of short fimbriae.¹ This part will be first expressed as a fusion protein with the N terminal fusion tag in the pET15b expression vector and the purified protein will subsequently be tested for binding with the selected candidate aptamers ( obtained from a later stage of ligand-basedSELEX) via ELISA-based binding assay.² Here the expression vector contains a "Thrombin site" which refers to a DNA sequence which contains the recognition site of thrombin (a protease). As a control experiment,after purification of the induced FimH2 protein carrying 6 X His affinity tag at its N-terminal end, thrombin enzyme will be used to cleave the His tag and the tag free protein will be again tested with the selected candidate aptamers ( obtained from a later stage of ligand-based SELEX) to validate if the aptamers are truly binding to the purified FimH2 protein and is showing no interaction with the His Tag.Thus by engineering this part we will be setting up a platform to purify His tag-free protein. For further information related to this part please visit our parts page FimH2 (BBa_K4449001)

• Staphylococcus epidermidis Bap like protein Bhp (Bhp) (BBa_K4449002)

This basic part encodes a virulence factor of S. epidermidis that helps in biofilm accumulation and is known as a surface adhesion protein or Bap (biofilm associated protein) that is commonly found in S. epidermidis strains.³ In our project this part will be expressed under the control of the T7 promoter of pET 28b+ expression vector. We will express this protein with a C terminal 6X His tag in BL21DE3 strain of E coli under IPTG induction and purify the protein using Ni NTA affinity column.

This part will be used for the negative selection round of ligand based SELEX and will serve as a negative control in our ELISA based binding assays. We have chosen this part encoding a virulence factor from Staphylococcus epidermidis because Staphylococcus epidermidis comprises a minority of the microflora of the lower part of the Urethra, and can be expected to be present as a contaminant in the urine cultures.⁴ This is an exclusively new part that has been added to the registry. For further information related to this part please visit our parts page Staphylococcus epidermidis Bap like protein Bhp (Bhp) (BBa_K4449002)

Primer parts

The following primer parts have been added to the Registry of Standard biological parts:

• pET28b FimH1 F: BBa_K4449003
• pET28b FimH1 R: BBa_K4449004
• pET15b FimH2 F: BBa_K4449005
• pET15b FimH2 F: BBa_K4449006
• pET28b Bhp F: BBa_K4449007
• pET28b Bhp R: BBa_K4449008

Generation of Aptamer sequences (BBa_K4449012)

Description

• Through this project we have generated a library of 500 random oligonucleotide sequences that have 40 random nucleotides in between flanked by a common forward and reverse primer sequence on both sides. These primers were added so that the selected sequence can be subjected to PCR amplification in the positive selection rounds of SELEX technique. Here in the 40 random nucleotide sequence a ratio of 3:2:2:3 is followed between A:T:G:C. The random oligonucleotide sequences were generated using python with the help of Jupyter Notebook.²

• Primer sequences:
  • Forward primer sequence: 5’-GTCTATATGATCTGTAACAC-3’
  • Reverse primer sequence: 5’-CCAGCAGTGAGTCATCAGAT-3’

Utilization

• The plan is to use this library to carry out multiple rounds of SELEX techniques to get a suitable nucleotide sequence that will effectively bind to our targeted proteins fIMH1 and FimH2.
• Here is the link to see the generated sequence: Click Here

Composite parts

The following composite parts have been added to the Registry of Standard biological parts:

• Apt4UTI C1: BBa_K4449009
• Apt4UTI C2: BBa_K4449010
• Apt4UTI C3: BBa_K4449011

• Improvement of an Existing Part

fimH HIS Tag (BBa_K3953004)

fimH HIS Tag (BBa_K3953004) is a DNA part that was developed by iGEM21_Pittsburgh (2021-10-21) which consists of the histidine tag sequence that codes for consecutive histidine amino acid residues within the fimH gene. Although this is an excellent tool in which the affinity of histidine tags for NiNTA beads will serve as a proof of concept for fimH binding, from literature surveys we found that an N or a C terminal His tag is more desirable for NiNTA based purification.⁶ In our project Apt4UTI, we have developed a platform to express C terminal and N terminal His tagged FimHprotein, since we think that putting a His tag at C terminal or N terminal end will be a better approach without interfering with the protein conformation and will ease purification process. The C terminal and N terminal His tag would enable proper folding of our protein of interest FimH1 and FimH2. FimH1 and FimH2 are the modified versions of the gene sequences derived from the plasmid pBAD-FimH-9x His (Addgene # 97305) which were developed for cloning into pET28 b+ (EMD Biosciences) and pET15b+ ( Novagen, EMD Millipore) respectively.

• Apt4UTI C1 (BBa_K4449009) will express our insert FimH1 with C terminal His tag.

• Apt4UTI C2 (BBa_K4449010) to express our insert FimH2 with N terminal His tag.

• In the construct Apt4UTI C2 (BBa_K4449010), the FimH2 will be expressed with a N terminal His tag along with a thrombin site, so that the enzyme thrombin can be used to remove the His tag in order to purify a tag free protein and validate that the aptamers are not showing any non-specific binding with the His tag.

Moreover with the help of our literature surveys we have devised several methodologies to measure our parts:

Methodologies to test our construct Apt4UTI C1 which will express C terminal 6X His tagged FimH1.

1. SDS PAGE:After purification of the 6X -His tagged protein, we will perform an SDS PAGE where we will run the molecular mass markers - 98, 64, 50, 36, 30, 16, 6, kDa (lane 1),Coomassie blue stained cytoplasmic soluble fraction preparation (lane 2), the purified FimH1-6X His protein ( lane 3) and the purified Staphylococcal Bap like Bhp protein-6X His (lane5 – negative control))¹. This will help us to validate our isolation and purification steps.
2. Receptor blots:Upon running the purified sample on an SDS PAGE the samples will be transferred to PVDF microporous membrane filters using a semi-dry blotting apparatus.Receptor blots of FimH1 6XHis and Bhp-6X His to α-D-mannosylated BSA (Sigma) will be then carried out by first incubating the filterblots with α-D-mannosylated BSA (0.5 mg l-1) followed by incubation with rabbit anti-BSA serum and finally with peroxidase-conjugated anti-rabbit serum to demonstrate the functionality of the FimH1 protein)¹, whereas Bhp is expected to show no interaction with the receptors that are specific for FimH1. Moreover the signal intensity of the obtained bands will be directly proportional to the concentration of our target protein.

3. ELISA based Binding assays - His-tagged protein (FimH1 -6X His) will be incubated with a biotin-labelled anti-His antibody, which will be immobilized in streptavidin-coated wells.² After incubation of FITC-labelled aptamers with protein-coated wells, HRPlabelled anti-FITC antibodies will be added. After washing, QuantaBlu Fluorogenic Peroxidase Substrate will be added as a readout of the aptamer-target binding and the fluorescence intensity is measured using a plate reader.² We would select those aptamer candidates which shows high intensity fluorescence signal upon interacting with Fim H1 and no or low signal intensity on interacting with Bhp .On the other hand we need to rule out those aptamers which emits positive signals upon interacting with Bhp.


Figure: Schematic illustration of ELISA-based binding assay. His-tagged protein is incubated with biotin-labeled anti-His antibody, which is immobilized to streptavidin coated wells. After incubation of FITC labeled aptamers with protein coated wells, HRPlabelled anti-FITC antibody is added. After washing, QuantaBlu Fluorogenic Peroxidase Substrate is added as a readout of the aptamer-target binding and the fluorescence intensity is measured using a plate reader. We expect an amplified positive signal for FimH 1-6X His and no signal for Bhp-6X His.(Adapted from Wang et al, 2019 Feb 30, Hum Gene Ther methods)

Methodologies to test our construct Apt4UTI C2 which will express N terminal 6X His tagged FimH2.

1. SDS PAGE:After purificationof the 6X -His tagged protein, we will perform an SDS PAGE where we will run the molecular mass markers - 98, 64, 50, 36, 30, 16, 6, kDa (lane 1),Coomassie blue stained cytoplasmic soluble preparation(lane 2), the purified FimH2-6X His protein (lane 3) and the purified Staphylococcal Bap like Bhp protein-6X His (lane5 – negative control).¹. This will help us to validate our isolation and purification steps.
2. Receptor blots:Upon running the purified sample on an SDS PAGE the samples will be transferred to PVDF microporous membrane filters using a semi-dry blotting apparatus.Receptor blots of FimH2 6XHis and Bhp-6X His to α-D-mannosylated BSA (Sigma) will be then carried out by first incubating the filterblots with α-D-mannosylated BSA (0.5 mg l-1) followed by incubation with rabbit anti-BSA serum and finally with peroxidase-conjugated anti-rabbit serum to demonstrate the functionality of the FimH2 protein¹, whereas Bhp is expected to show no interaction with the receptors that are specific for FimH2. Moreover the signal intensity of the obtained bands will be directly proportional to the concentration of our target protein.

3. ELISA based Binding assays: His-tagged protein (FimH2 -6XHis) will be incubated with a biotin-labelled anti-His antibody, which will be immobilized in streptavidin-coated wells. After incubation of FITC-labelled aptamers with protein-coated wells, HRPlabelled anti-FITC antibodies will be added.² After washing, QuantaBlu Fluorogenic Peroxidase Substrate will be added as a readout of the aptamer-target binding and the fluorescence intensity is measured using a plate reader.² We would select those aptamer candidates which shows high intensity fluorescence signal upon interacting with FimH2 and no or low signal intensity on interacting with Bhp .On the other hand we need to rule out those aptamers which emits positive signals upon interacting with Bhp.


Figure: Schematic illustration of ELISA-based binding assay. His-tagged protein is incubated with biotin-labeled anti-His antibody, which is immobilized to streptavidin coated wells. After incubation of FITC labeled aptamers with protein coated wells, HRPlabelled anti-FITC antibody is added. After washing, QuantaBlu Fluorogenic Peroxidase Substrate is added as a readout of the aptamer-target binding and the fluorescence intensity is measured using a plate reader. We expect an amplified positive signal for FimH 2-6X His and no signal for Bhp-6X His. ( Adapted from Wang et al , 2019 Feb 30, Hum Gene Ther methods)

Documentation of an existing part

(BBa_K1104102)

In our project Apt4UTI we will be expressing FimH protein which is a major component of the Type 1 pilli that specifically helps the uropathogens to adhere to a spectrum of alpha-D-mannosides found in mammalian tissue surface.

For this we have developed two constructs Apt4UTI C1 and Apt4UTI C2 which will be expressing 6X his tagged FimH protein. Our team went through countless literature surveys in order to find out a suitable methodology to test the functionality of our purified 6X His tagged Fim H.

We found that Team iGEM13_NYMU-Taipei devised a fluorescence based methodology to test the functionality of the pLac+RFP-FimH protein by expressing it as a fusion protein of RFP-FimH and then checking the FimH binding to mannose. Later From our literature surveys related to the testing of FimH protein, we have found another in vitro methodology that can serve as a functionality check of purified FimH binding to mannose which used alpha-D mannosylated BSA (mimicking the mannose receptor) as a matrix for FimH binding .The technique is termed as Receptor Blot and involves:

1. SDS PAGE of periplasmic preparations of purified proteins
2. Transfer to PVDF microporous membrane filters
3. Incubation with alpha-D mannosylated BSA.
4. Incubation with rabbit anti-BSA serum and finally with peroxidase-conjugated anti-rabbit serum.

For further details regarding the methodology visit pLac+RFP-FimH(BBa_K1104102)

References:

1. Schembri, M.A., Hasman, H. and Klemm, P; Expression and purification of the mannose recognition domain of the FimH adhesin; 01 July 2000; FEMS Microbiology Letters, 188(2), pp.147–151. DOI:10.1111/j.1574-6968.2000.tb09186.x
2. Tao Wang , Wang Yin,Hadi AlShamaileh, Yumei Zhang, Phuong Ha-Lien Tran , Tuong Ngoc-Gia Nguyen, Yong Li, Kuisheng Chen, Miaomiao Sun, Yingchun Hou, Weihong Zhang, Qingxia Zhao, Changying Chen, Pei-Zhuo Zhang, and Wei Duan; A detailed protein-SELEX protocol allowing visual assessments of individual steps for high success rate, 2019 Feb30, Human Gene Therapy MethodsVol. 30, DOI:10.1089/hgtb.2018.237
3. Amirmorteza ,EbrahimzadehNamvar,Sara Bastarahang,Niloufar Abbasi,Ghazaleh Sheikhi,Ghehi ,Sara,Farhadbakhtiarian,Parastoo Arezi,Mahsa Hosseini,Sholeh Zaeemi Baravati, Zahra Jokar,Sara Ganji Chermahin ,Clinical characteristics of Staphylococcus epidermidis: a systematic review ,2014 Sep 30, GMS Hygiene and Infection Control, Vol. 9(3), DOI: 10.3205/dgkh000243
4. Shankar Upadhyayula, Mamatha Kambalapalli, and Basim. Asmar, Staphylococcus epidermidis Urinary Tract Infection in an Infants;24 July 2012,Case Reports in Infectious Disease,Volume 2012, Article ID 983153. DOI: 10.1155/2012/983153
5. pBAD-FimH-9xHis; Adgene #97305 https://www.addgene.org/97305/
6. His tagged Proteins; Creative biostrucrure https://www.creative-biostructure.com/his-tagged-proteins-553.htm