iGEM — Results

Results

Experimental Results:

For an aptamer based detection kit for Urinary Tract Infections, we started with two approaches, Whole cell based and ligand based approach. The plan made out for a whole cell based approach did not meet the facilities available in the institute currently. The team decided to shift to a ligand based approach. However, IISER berhampur being in the rural area, there were delays in ordering and transportation which slowed the process. With the best pf our capabilities we performed the experiments and the results obtained till now are as follows:

DH5 Alpha Plasmid:

Cells transformed with pET28b+ plasmid, kindly provided by iGEM team CODEM, IISER Berhampur, were inoculated into LB media.

pET28b+ plasmid isolation

Figure: Lane 1 & 2 represent plasmids from the QIAGEN Kit and Lane 3 is plasmid from Promega

Plasmid was isolated from transformed cells using different methods. Alkaline lysis for plasmid isolation created purity issues. Therefore, the team decided to shift to kit based methods. Promega kit and QIAGEN were then used to perform the analysis. We got the highest concentration of plasmid when we used the QIAGEN kit and this was mainly used for further experiments.Through Alkaline lysis method we got very less concentration but through the gel electrophoresis it was confirmed that there was no genomic DNA contamination. The plasmid produced results are shown here.

Concentration of the plasmids obtained from different methods using nandrop is shown here:

QIAGEN Kit

Colony	Concentration (ng/μl)	A260/A280	A260/A230
C1	87.2	1.98	2.10
C1 (Replicate)	73.2	1.99	2.02

Promega Kit

Colony	Concentration (ng/μl)	A260/A280	A260/A230
C1	28.8	1.77	1.79
C1 (Replicate)	30.1	1.80	1.72

Alkaline Lysis

Colony	Concentration (ng/μl)	A260/A280	A260/A230
C1	31	1.87	2.20

FimH fragment PCR gel electrophoresis:

For the cloning experiment, the FimH fragments were amplified using polymerase chain reaction. For the optimization, annealing temperature was kept at 58 degree celsius. After the amplification, the PCR product and isolated plasmid were digested using appropriate restriction enzymes. Gel bands and concentration obtained as a result are shown below:

PCR amplified FimH insert

ds DNA	Concentration (ng/μl)	A260/A280	A260/A230
FimH Insert	58.1	1.70	0.19

Digested Plasmid and Insert. Plasmid is of 5368 bps and insert is of 557 bps.

ds DNA	Concentration (ng/μl)	A260/A280	A260/A230
pET28+	19.3	1.85	0.01
FimH	34.2	1.61	0.14

After the restriction digestion, digested products were extracted and used for ligation. Ligated products were then used for transformation of DH5 alpha colonies. For the confirmation of ligated plasmids the colonies were used to perform Colony PCR. We are still in the process of optimization of the purification process.

Transformed colonies of Ligated Product

Map of restriction digestion of Xho Ⅰ and Nco Ⅰ and ligation using snapgene

Cloning

For the cloning experiment we have developed three plasmids using Snapgene. down below are shown the plasmids designed by the biobrick team for cloning experiments:

Figure: This plasmid is obtained from the cloning of FimH into pET28+ for the expression of FimH protein.

Figure: This plasmid is for the assurance that the aptamer does not bind to the his tag instead binds to the FimH protein only.

In future planned experiments, the cloned plasmid and transformed cells we can now initiate the process of purification of FimH protein using Ni-NTA Kit. Which will be then used for the ligand based SELEX to find the aptamer high specific binding affinity towards the fimH protein and which will then be validated by the whole cell selex.

Figure: This plasmid is for the negative control to ensure that the aptamers don't bind to protein other than FimH.

Modelling Work

We generated the 3D conformation of aptamer sequences(ssDNA sequences) using a similar protocol used for RNA sequence structure prediction.
We also generated the 3D structure of FimH sequence using AlphaFold2 deep learning model Through our dry lab work, we screened aptamers with highest affinity for ligand FimH. After the purification of the protein this in-silico generated library will be used for the screening of the best aptamer for usage in the detection kit.
Using the end product from aptamer assay, we intend to develop the UTI detection kit.

3D structure of an Aptamer from our Aptamer library

Project beyond the detection kit

The target of our project of building a detection kit doesn't stop there, we on a small level in rural areas of Odisha tried to spread awareness about UTI so people become aware of the disease first. We as a team then spread the word in regional language so that the taboo of talking about UTI in public could be demolished. Only once rurals will talk about this infection, will they come forward to use a device like a Detection kit.
We held an awareness campaign to spread the word of usage of detection methods, current methods of detection and precautionary measures that can be taken even in underdeveloped villages with minimal or no sanitary conditions.
In our efforts we conducted an awareness campaign and generated a survey which would help us to lead our further research.
As a little step we proposed to build public toilets and water supply to villages lacking basic sanitation, for which we are seeking out funds.
With the information obtained from the survey we tried our research to be based on their underdeveloped condition where a laboratory and expertise can’t be managed easily.