Test PCR for FASTmiR-146a-D1

Aim To optimise PCR conditions for Scale-Up PCR for FASTmiR-146a-D1

Procedure Prepared 15 μl reaction with varied concentrations of FASTmiR-146a-D1 template; 0 ng/μl, 2.5 ng/ μl, 5 ng/μl, 10 ng/μl.

Results Found 10 ng/μl to be the ideal concentration of the template.

Figure 1. Test PCR of FASTmiR-146a-D4.

Labels Lane 1: 100 bp DNA Ladder, Lane 2: 0 ng/μl negative control, Lane 3: 2.5 ng/μl template, Lane 4: 5 ng/μl template, Lane 5: 10 ng/μl

ConclusionAfter few trials, we found for the working concentration of 10 ng/μl of template the following PCR conditions

Test PCR for FASTmiR-146a-D2 to D5

Aim To test the length of FASTmiR-146a-D1, D2, D3, D4, D5 and optimise its PCR conditions.

Result Found the length of the designs to be approx. 200 to 210 bps.

Conclusion Expected length for the designs achieved. 

Results Found 10 ng/μl to be the ideal concentration of the template.

Figure 2. Test PCR for all designs of FASTmiR-146a.

Labels Lane 1: 100 bps DNA ladder, Lane 2: 0 ng/μl template negative control, Lane 3: FASTmiR-146a-D1, Lane 4: FASTmiR-146a-D2, Lane 5: FASTmiR-146a-D3, Lane 6: FASTmiR-146a-D4, Lane 7: FASTmiR-146a-D5.

Test PCR for FASTmiR-27a and FASTmiR-30c Designs:

Aim To test the lengths of our FASTmiR-27 and FASTmiR-30c designs

Results Found FASTmiR-27a-D1 and D2 to be approx. 190 bps and the rest loaded designs to be of expected length of approx. 140 bps.

Figure 3. Test PCR for all designs of FASTmiR-27a and FASTmiR-30c

Labels Lane 1: 100 bps DNA ladder, Lane 2: FASTmiR-27a-D1, Lane 3: FASTmiR-27a-D2, Lane 4: FASTmiR-27a-D3, Lane 5: FASTmiR-30c-D1, Lane 6: FASTmiR-30c-D2, Lane 7: FASTmiR-30c-D3.

IVT Confirmation for FASTmiR-146a

Aim To check the expected length of the IVT product for FASTmiR-146a-D5

Results Found the IVT to be of expected length of 120bps.

Figure 4. IVT product for FASTmiR-146a-D5

Labels Lane 1: IVT product 3 μl loaded, Lane 2: IVT product 1 μl loaded, IVT product 0.5 μl loaded

Fluorescence Test of miR-146a-D4

 

Figure 5.Fluorescence readout for miR-146a-D4 after 30 mins incubation.

Figure 6. Table explaining the fold change for fluorescence readout for miR-146a-D4 after 30 mins incubation.

 

Figure 7. Fluorescence readout for miR-146a-D4 after 1 hour incubation.

Figure 8. Table explaining the fluorescence readout for miR-146a-D4 after 1 hour incubation.

 

Fluorescence Test of miR-222-D2 

Figure 9. Fluorescence readout for miR-222-D2 after 30 mins incubation.

 

 

Figure 10. Table explaining fluorescence readout for miR-222-D2 after 30 mins incubation.

Figure 11. Fluorescence readout for miR-222-D2 after 1 hour incubation.

Figure 12. Table explaining fluorescence readout for miR-222-D2  after 1 hour incubation.

Testosterone Detection

Annealing of Trigger-Aptamer & Trigger-Target

Aim To confirm the annealing for the formation of trigger-T6 aptamer complex

Procedure Equimolar concentrations (50 μM) of trigger and aptamer for the trigger-aptamer complex in 1x TMS (Tris magnesium sulfate) buffer and DEPC-treated water.

Results:  Fig 1. indicates significant trigger-aptamer annealing for all three testosterone designs, as shown in lanes 5, 6, and 7. The shift in the size of bands on Native PAGE indicates the annealing of the single-strand sequence, as the binding of two strands would increase the complex's molecular weight.

 

Testosterone Detection: Fluorescence Assay & In-vitro transcription of Broccoli

 

Aim To detect various concentrations of testosterone via fluorescence assay for Approach 2: dual aptamer without phi29 DNA polymerase (T6-design 3; T6-D3; Trigger-BBa_K4438605; Target-BBa_K4438606)

Procedure Incubate 10 μM of annealed trigger-T6 aptamer (D3) with 0 μM, 1 μM, 10 μM, and 100 μM of ethanol-dissolved testosterone propionate in 1x binding buffer and 1 μM target-design 3, at 25℃ celsius for 1 hour. 

This is followed by in-vitro transcription by T7 RNA polymerase of the target strand to produce Broccoli aptamer for different testosterone concentrations.

Results  For T6-D3, the fluorescence assay showed no significant difference in fluorescence at increasing testosterone concentrations. This indicates that the trigger has more affinity for the target than the aptamer due to the number of complementary sequences between them. 

Figure 2. Fluorescence readout for T6-D3 at various concentrations of testosterone

Comment  Here, we realized that the range of concentration for the biomarker is too narrow for the first trials. Hence, we used higher concentrations for the test of further designs. 

Aim To detect various concentrations of testosterone via fluorescence assay for Approach 1: dual aptamer with phi29 DNA polymerase. (T6-Design 1; T6-D1; Trigger-BBa_K4438601; Target-BBa_K4438602 and T6-Design 2; T6-D2; Trigger-BBa_K4438603; Target-BBa_K4438604)

Procedure 

Incubate 10 μM of annealed trigger-T6 aptamer (D1 and 2) with 0 μM, 10 μM, 100 μM, and 1 mM of ethanol-dissolved testosterone propionate in 1x binding buffer and 1 μM target-design 3, at 25℃ celsius for 1 hour. 

The hormone-incubated products are incubated with phi29 DNA polymerase for the extension of the 3’-5’ trigger strand to produce duplexed DNA. This step is crucial for in-vitro transcription to take place

This is followed by in-vitro transcription by T7 RNA polymerase of the target strand to produce Broccoli aptamer for different testosterone concentrations.

Results 

1. T6-Design 1: The fluorescence readout (Fig 3) indicates that the fold change in the fluorescence is not significant. Hence, there is no significant detection of testosterone.
2. T6-Design 2: The fluorescence readout (Fig 3) indicates that the fold change in the fluorescence is significant of 4 folds for 1mM of testosterone. This result shows that T6-D3 

Figure 3.  Fluorescence readout for T6-D 1 and 2 at various concentrations of testosterone

 

Figure 4.  Table for fluorescence readout for T6-D1

Figure 5. Table for fluorescence readout for T6-D2 

 

Comment: Although T6-D2 shows 4 fold increase in fluorescence and detect 1mM of Testosterone, we believe that the following optimizations can lead to higher sensitivity i.e detection of lower levels of testosterone:

1. T6 aptamer - Trigger 3 relative concentrations while annealing 
2. Tweaking few bases of the trigger to allow release of trigger at lower concentrations

Progesterone Detection

Annealing of Trigger-Aptamer & Trigger-Target

Aim  To confirm the annealing for the formation of trigger-P4G13 aptamer complex

Procedure  Equimolar concentrations (50 μM) of trigger and aptamer for the trigger-aptamer complex in 1x TMS (Tris magnesium sulfate) buffer and DEPC-treated water.

Results   Fig 6, Lane 5 indicates the annealing of Trigger 4- P4G13 aptamer

     

Verification of release for Trigger upon incubation with Progesterone

Aim To verify release of trigger upon incubation with Progesterone

Procedure

1. Incubate 10 μM of annealed trigger-P4G13 aptamer (Design 4) with 0 μM, 1 μM, 10 μM, and 100 μM of ethanol-dissolved progesterone in 1x binding buffer and 1 μM target-design 4, at 25℃ celsius for 1 hour. 
2. Incubate 10 μM of annealed  trigger-P4G13 aptamer (Design 4) with 0 μM, 10 μM, 100 μM, and 1000 μM of ethanol-dissolved progesterone in 1x binding buffer and 1 μM target-design 4, at 25℃ celsius for 1 hour. 

Results  

From Fig7 and 8, we can observe that a fraction of Triggger 4 is unbound from the P4G13 aptamer, irrespective of  the concentration of Progesterone. 

However, Fig 8 indicates that the binding of P4G13 aptamer and Progesterone increases upon increasing hormone concentration. We speculated the free triggers from here could increase the background fluorescence after in-vitro transcription.

Figure 7. 16% Native -PAGE: Test for Trigger 4 release upon incubation with Progesterone

 

Lane 1:Trigger (Design 1); Lane 2: Progesterone Aptamer; Lane 3: Aptamer + Trigger (Progesterone= 0 μM), Lane 4: Aptamer + Trigger (Progesterone= 1 μM); Lane 5: Aptamer + Trigger (Progesterone= 10 μM); Lane 6:  Aptamer + Trigger (Progesterone= 100 μM) 

Incubation with different concentrations of Progesterone

Figure 8. 20% Native-PAGE:Test 2 for Trigger 4 release upon incubation with Progesterone

Lane 1: Size Standard (66 Nucleotides); Lane 2: Aptamer; Lane 3:Aptamer + Trigger , Lane 4:  Aptamer + Biomarker ( 10 μM); Lane 5:Aptamer + Biomarker ( 100 μM); Lane 6: Aptamer + Biomarker ( 1000 μM); Lane 7: Aptamer + Trigger + Biomarker (10 μM); Lane 8: Aptamer + Trigger + Biomarker (100 μM); Lane 9: Aptamer + Trigger + Biomarker (1000 μM)

Verification of extension of DNA strand by phi 29 polymerase

Aim: To verify extension of annealed DNA stands by phi 29 polymerase

Procedure

1. For a reaction volume of 20ul, add 0.5 uL Phi29 polymerase, 5 uL annealed DNA, 2 uL dNTPs, 2 uL Bovine Serum Ablumin (BSA), 2 uL 10xphi29 polymerase buffer,8.5 uL autoclaved milliQ water in a PCR tube. 
2. Incubate at 30 degrees celsius for 30 minutes and then 65 degrees for 10 min. 
3. Verify using Native PAGE. 

Results 

Figure 9. indicatesthe extension of trigger4 and target4 by phi29 polymerase. However, as predicted we observed extension in 1000 μM concentration of progesterone,but unfortunately we could not visualise the triggers due to their length. 

Progesterone Detection: Fluorescence Assay & In-vitro transcription of Broccoli

Aim To detect various concentrations of progesterone via fluorescence assay for Approach 1: dual aptamer with phi29 DNA polymerase (P4G13-Design 4; P4G13-D4; Trigger-BBa_K4438707; Target-BBa_K4438708)

Procedure 

Incubate 10 μM of annealed trigger-P4G13 aptamer (D4) with 0 μM, 10 uM, 100 μM, 1 mM and 10 mM  of ethanol-dissolved progesterone in 1x binding buffer and 1 μM target-design 3, at 25℃ celsius for 1 hour. 

The hormone-incubated products are incubated with phi29 DNA polymerase for the extension of the 3’-5’ trigger strand to produce duplexed DNA. This step is crucial for in-vitro transcription to take place.

This is followed by in-vitro transcription by T7 RNA polymerase of the target strand to produce Broccoli aptamer for different testosterone concentrations.

Figure 10. Fluorescence readout: P4G13-D4 - 1

Results No significant increase in fluorescence upon increasing concentration of progesterone. Hence, we understand that this design could potentially work only after optimising and trying variations of P4G13 Trigger 4.

 

Aim To test trigger- target interaction via in-vitro transcription and effect on various concentrations of trigger on fluorescence readout (P4G13-Design 4; P4G13-D4)

Procedure 

Incubate 50 μM of target(D 4) with annealed with 10 μM, 20 μM, 30 μM, 40 μM and 50 mM trigger concentration in 1x TMS buffer for 1.5 hrs

The hormone-incubated products are incubated with phi29 DNA polymerase for the extension of the 3’-5’ trigger strand to produce duplexed DNA. This step is crucial for in-vitro transcription to take place.

This is followed by in-vitro transcription by T7 RNA polymerase of the target strand to produce Broccoli aptamer for different testosterone concentrations.

Figure 11. Fluorescence readout: P4G13-D4 - 1

Results We observed slight increase in fluorescence upon changing trigger-target concentration ratio. Hence, we understand that this design could potentially work only after optimising and trying variations of P4G13 Trigger 4.

Quinine and C-Reactive Protein Detection

Annealing of Trigger-Aptamer

Aim  To confirm the annealing for the formation of triggers-MN4 aptamer complex

Procedure  Equimolar concentrations (50 μM) of trigger and aptamer for the trigger-aptamer complex in 1x TMS (Tris magnesium sulfate) buffer and DEPC-treated water.

Results Fig 12 indicates that Design 5 and 6  for MN4 aptamer did not show any significant annealing of MN4 aptamer-Trigger.

Verification of release for trigger upon incubation with Quinine

Aim To verify release of trigger upon incubation with Quinine

Procedure Incubate 10 μM of annealed trigger-MN4 aptamer (D3) with 0 μM, 1 μM, 10 μM, and 100 μM of DMSO-dissolved quinine hemisulfate salt monohydrate in 1x binding buffer and 1 μM target-design 3, at 25℃ celsius for 1 hour. 

Results  Fig 13 indicates that there is no significant release of trigger 3 even at high concentration of quinine. Hence we speculate that this trigger/aptamer could be tweaked to detect this

Comments 

1. None of our native-PAGE assays could indicate annealing of the trigger-aptamer. So we went back to our in-silico analysis and picked Design 3 to test its incubation with quinine and test one of our designs       due to limitations of time.

2. We could not test designs for C-reactive protein due to delay in delivery of the ordered protein.

Conclusion

We understood that the method of combining in-silico and hit and trial experimental design involves high throughput and time consuming experiments. This applies especially for our dual aptamer approach.  However, in the limited time and lab resources we had access to, we were able to successfully characterize a design to detect testosterone (T6; BBa_K4438600) at 1mM concentration. The experiments and designs can be easily optimized to improve the sensitivity further.

Protein Purification

PROLIDASE

Aim To purify histidine-tagged prolidase from the Rosetta strain. There are no existing DNA aptamers, and prolidase is required to develop DNA aptamers against it using SELEX. 

Protein expression and purification

First, we expressed the prolidase in 20 mL 2xYT (supplemented with 100 ug/mL ampicillin and 34 ug/mL chloramphenicol) before proceeding with the bulk. 

Protein expression was induced with 1mM IPTG (link to protein purification protocol). After purification using Ni-NTA affinity chromatography, we ran SDS-PAGE to confirm the presence and purity of our protein of interest.

Lane 9 shows a faint band between 51-70 kDa range which is our expected range as the molecular weight of prolidase is 54 kDa. This confirms the presence of prolidase in elute fraction.

Next, we proceeded with the 2L bulk culture. A similar protocol was followed for this. Given below is the SDS-PAGE gel image for washes and elute fractions.

After concentrating and desalting the elute fractions, the nanodrop concentration obtained was 6.8295 mg/mL.

IRISIN

Aim To purify histidine-tagged irisin from E.coli BL21(DE3) strain.

Plasmid DNA isolation 

Isolation of the plasmid construct pET-15b-irisin.

Nanodrop concentration- 47.65 ng/μL.

Preparation of competent cells and transformation: The plasmid construct pET-15b-irisin was transformed into Escherichia coli BL21 (DE3) cells.

Protein expression and purification

From the freshly transformed plate, we picked 6-7 colonies and inoculated 20 mL of LB medium with 100 ug/mL of ampicillin. We induced the expression of protein with 0.5 mM IPTG followed by cell lysis and purification with Ni-NTA column chromatography (link to protein purification protocol page)

We ran SDS-PAGE with different fractions of protein obtained to confirm the presence and purity of irisin.

Figure 4. SDS-PAGE result of purification of irisin from the supernatant. Lane 1:10-315 kDa protein ladder; Lane 2: Uninduced cell pellet; Lane 3: Induced cell pellet, Lane 4: Lysis supernatant; Lane 5: Lysis pellet; Lane 6: Flow-through after binding; Lane 7: Wash 0 (10mM imidazole); Lane 8: Wash 1 (50 mM imidazole) and Lane 9: Elute (300 mM imidazole)

We observed a band between 10-26 kDa range in lanes 4, 6, 7, 8, and 9. 

Conclusion Papers report different molecular weights of irisin. We suspect that the clear band obtained corresponds to irisin.

SELEX

VERIFICATION OF THE ORDERED DNA LIBRARY AND THE PRIMERS

Aim To verify the ordered DNA library and primers for its length and purity

Procedure 5 μl of 10 μM samples loaded in 16% Urea page, run for 3 hours at 120 volts.

Results Found that the given sample of DNA Library is not of preferred length.

Inference 

1. Although we order a DNA library of 79 nucleotides, we received a faulty sequence of approximately 20 nucleotides. 
2. The library is not suitable to carry forward with incubation of protein.

VERIFICATION OF THE SYNTHESISED DNA LIBRARY 

Aim To verify the synthesized DNA library for its length and purity

Procedure 5 μl of 10 μM samples loaded in 16% Urea page, run for 3 hours at 120 volts.

Results Found that the given sample is of preferred length(79 neuleotides)

Inference

The synthesised library is pure and is at the correct position with respect to the size standard.

PCR BASED VERIFICATION OF THE SYNTHESIZED LIBRARY

Aim to amplify and verify the primers and the library

Procedure 

1.5 μl of the synthesized DNA library was mixed in a 25 μl PCR reaction. 5 μl of 10 times diluted PCR sample was loaded into 10% native page for verification.

Gel was run for 60 minutes at 120 volts.

Results the PCR amplification works well with given library and the set of primers.

Inference

1. The library has the required sequences.
2. Primers function optimally.
3. The duplexed library can be used for incubation with protein followed by asymmetric PCR.

Conclusion The synthesized library is of good quality and can further be used. 

INTERLAB

Calibration of 96-well plate

AimTo calibrate the 96-well plate.

Result The calibration of the 96-well black-sided costar plate was done and the readout came out as attached. https://static.igem.wiki/teams/4438/wiki/wetlab-results/interlab-calibration-iiser-tirupati-india-2022-sheet1.pdf

Some of the wells had an absorbance that was greater than detectable by the instrument available to us. This could be because of the dilution error in the calibration protocol that was provided by the Engineering Committee during the initial phase, the protocol for which was later modified by them.

Transformation of Escherichia coli DH-5𝛼 with Test devices

Multiple attempts at transformation failed and the similar issue faced by different teams suggested a fault in the plasmid provided in the distribution kit.

We speculate that this could be because of

1. Plasmid-strain incompatibility
2. Low concentration of plasmids
3. Issues with the resuspension protocol provided

Conclusion

Due to time constraints and the inability to devise a strategy around the allegedly defective plasmid, our experiments for the Interlab study were halted. Although we would have loved to complete the experiments.