The results outline the experimental work carried out over 1-2 months of lab work. First, we describe the construction and characterization of the various sensing modules cloned in the E-coli strain using Snapgene. To separate the DNA fragments according to size and charge after a restriction digest, the team simulated agarose gel electrophoresis in SnapGene. The second part presents the preliminary experiments showing the digestion and ligation of our parts. With lots of research and iterations, the team designed the genetic parts for our project in silico with the SnapGene application and sent them out for synthetization. Our less experienced team with biological lab work (70% of the team are computer science, mechanical and electrical engineering majors) started practicing in the lab with digestions, ligations, transformations, gel electrophoresis, and making competent cells. We also explain the troubleshooting of these initial experiments determining the issues with the ligation and digestion. Then, we explain the corrections of the digestion and ligation protocols resulting from this troubleshooting. Also, we present the results obtained from the final try, continued with miniprep, PCR and gel electrophoresis to characterize the part. And finally, the result for the first try of the hydrogel encapsulation and some proposed future experiments to obtain the expected results.
Before performing this experiment in the Lab, the team utilized Snapgene to simulate the digestion and ligation of the various parts. In this approach, we first performed a linear ligation between the sensing module and kill switch fragments (Au and UV, As and UV, Fe part 1 & 2 and UV) to evaluate if it's feasible to be ligated. This was done successfully, as represented in figures (1a,2a, and 3a). We used restriction and insertion cloning to insert these fragments into the plasmid (pUC 19). By inserting, we cut the plasmid with the EcoR1 and Pst1 to create sticky ends to facilitate ligation. We also cut the fragments with EcoR1 and PSt1 to maximize ligation efficiency. This resulted in complete plasmids with various sensing module inserts, as shown in figures 1b, 2b, and 3b. Figure 1c. 2c and 3c depict the vectors and inserted fragments with their various sizes (base pairs), which enabled the team to visualize how the insertion was done and the size of the various components.
1. Au and Kill Switch Model
Figure 1a: Linear ligation of gold inducer and the kill switch to create a complete gene insert
Figure 1b: Ligated plasmid with Au-UV insert
Figure 1c: Illustration depicting the vector (pUC 19) and the fragment (Au inducer-UV)
2. As and Kill switch Module
Figure 2a: Linear ligation of arsenic inducer and the kill switch to create a complete gene insert
Figure 2b: Ligated plasmid with As-UV insert
Figure 2c: Illustration depicting the vector (pUC 19) and the fragment (As inducer-UV)
3. Fe and Kill switch Module
Figure 3a: Linear ligation of ferric inducer and the kill switch to create a complete gene insert
Figure 3b: Ligated plasmid with As-UV insert
Figure 3c: Illustration depicting the vector (pUC 19) and the fragment (Ferric inducer-UV)
In this simulation, the team wanted to identify the inserted fragments that bear the inducers or sensing modules. From the ligation and cloning, the expected sizes for Fe, Au and As-UV fragments were 2455bp, 2368bp and 2324bp, respectively. These sizes were checked with the 1% agarose gel electrophoresis, where the NEB 1 kb DNA ladder was used for the simulation to quantify the inserts and the DNAs. From figure 4, using the ladder marker at 3.0kb, we obtained the expected size as shown. From here, we concluded that since the simulation works, we could go ahead and do the lab experiments to confirm what is expected from the simulations.
Figure 4: Identifying fragments that bear the inserted genes (Au, Fe and As inducers) by gel electrophoresis simulation.
Once the parts arrived, we went ahead with digestion of our genetic parts or genes of interest (Gold [Au], Arsenic [As] and Iron [Fe (I & II)] sensing modules, and the autolytic UV kill switch modules) and the vector we chose for our design (Puc19 Plasmid). We continued with ligating the different gene fragments with the vector and performed transformation with the chassis we chose (Escherichia coli). While doing these experiments, we also made competent cells from bacteria culture that were grown overnight.
Figure 5: Infographics showing the experimental processes that were undertaken
1.1 DIGESTIONWe made 25 ul solutions for each digestion. We made three different tubes of digested puc19 DNA parts, three different tubes with digested Gold, Arsenic, and Iron (I), and three different tubes with two digested UV kill switch modules and an Fe (II) genetic part. After these digestions were conducted, we went ahead with ligating the various corresponding parts with the digested vector.
After these digestions were conducted, we went ahead with ligating the various corresponding parts with the digested vector.
1.2 LIGATIONWe made 25 ul solutions for each digestion. We made three different tubes of digested puc19 DNA parts, three different tubes with digested Gold, Arsenic, and Iron (I), and three different tubes with two digested UV kill switch modules and an Fe (II) genetic part. After these digestions were conducted, we went ahead with ligating the various corresponding parts with the digested vector.
After ligation, we transformed the parts from the three tubes from ligation with the competent cells and allowed for overnight growth.
1.3 OBSERVATION, RESULTS AND TROUBLESHOOTAfter the overnight growth, we did not observe any growth or colonies on our agar plates for Arsenic and Fe sensing modules. However, we observed very few colonies for the Au sensing module. We took the colony and added it to ampicillin media, added some gold particle-chippings and grew it overnight. We plated the culture with the gold particles on ampicillin plates to observe growth and color change over a period. No color change was observed for this investigation, however. We then suspected something might have been wrong with the concentration of the plasmid used. We suspected the plasmid might have been over diluted to undergo digestion and ligation. Thus, we sought to find out if our suspicion was correct. We performed a digestion, ligation, and transformation for only the puc19 plasmid to obtain a very concentrated plasmid after we conducted a mini prep. After the mini prep, we ran a gel, to confirm if we were correct. From the gel image shown below we were correct. The second well which had the initial diluted plasmid DNA had no trace of DNA on its column, while the next four wells which had the concentrated plasmid had traces of DNA on their columns.
Figure 6: Gel electrophoresis results comparing two different plasmids obtained with different miniprep kits to the NEB 1kb MW ladder
Another suspicion we had about the results we had was whether the mini-prep kit we used was problematic. Thus, for the gel image shown above, we conducted two mini-prep with an old and a new kit. The columns labelled A1 and A2 were minipreps conducted with the new kit and the columns labelled B1 and B2 were minipreps conducted with the old kit. We had two columns for each kit used because we used two different colonies for each kit. Based on the results from the gel, we concluded that both our old and new mini-prep kits had no issues.
1.4 PLASMID CONCENTRATION TROUBLE SHOOTWe were able to troubleshoot what was wrong and realized the plasmid DNA we used initially was very diluted. After obtaining the concentrated plasmid DNA, we went ahead to ascertain what concentration of the current plasmid DNA used would be ideal for our experiments. We diluted the current concentrated plasmid DNA into three concentrations (1:2 concentration, 1:5 concentration and a 1:10 concentration). We ran a gel and based on the results from the gel image below, we chose to go with a 1:10 concentration for our subsequent experiments.
Figure 7: Gel electrophoresis results comparing different pUC 19 ratios
1.5 DIGESTION AND LIGATION TROUBLE SHOOT
The initial results obtained also raised concerns about whether we had issues with the digestion and ligation of our parts. Thus, before we went ahead with the second lab experiment with the remaining of our synthesized parts, we wanted to ascertain if our digestion and ligation worked with no errors. As a result, we performed digestion and ligation with the 1:10 concentrated plasmid DNA from the previous experiment. We digested with the restriction enzymes, SpeI and XbaI since both when used to cut the vector would have sticky ends that are compatible. For this experiment, we used two different approaches to digestion. One digestion approach involved making a Mastermix made up of the reagents used in digestion and the other approach involved no Mastermix. We suspected that making a Mastermix before digestion might further dilute the concentrations of the restriction enzymes and the DNA parts. Thus, we conducted these experiments to make sure our suspicions were correct or otherwise.
For this experiment, we had four digestions of pUC 19. Two of these digestions were done using the Mastermix approach (MM) and the other two were digested using the No Mastermix approach (No MM). One of the digested Puc19 part tubes for each digestion approach were transformed directly while the other two for each digestion approach went ahead to ligation.
We figured out that, if digestion worked then, the transformation for the digested parts from each approach should have no growth when plated on the ampicillin agar plates. The reasoning behind this is that, if the plasmids were not correctly cut or digested, the bacteria would have full plasmids with ampicillin resistance which would mean that these bacteria would not be killed by the ampicillin antibiotic. However, if they were cut correctly, the bacteria would not have ampicillin resistance from the plasmid DNA and would be destroyed by the ampicillin antibiotic from the agar plate and thus would experience no growth.
We further ligated the other two tubes, one each from the two digestion approaches, transformed with competent cells, and allowed for growth overnight. We had two control experimental setups for this experiment:
➕ Positive control (transformed E. coli bacteria with undigested and unligated Puc19 plasmid)
➖ Negative control (transformed E. coli bacteria only)
The positive control is expected to have growth because the E. coli bacteria would have ampicillin resistance from the plasmid DNA and would be destroyed by the ampicillin antibiotic from the agar plates. The negative control however is expected not to have any growth since it has no plasmid with ampicillin resistance and will be destroyed by the ampicillin antibiotic from the agar plate.
Figure 8: results obtained from Digestion and ligation troubleshoot (a) transformed digested plasmid with master mix (b) transformed digested plasmid without master mix (straight NEB protocol) (c) Transformed ligated plasmid with master mix (d)Transformed ligated plasmid with no master mix (e) positive control (pUC 19 + competent cells) (f) Negative control (Just competent cells with no plasmid or DNA)
From the results we obtained after transformation as seen above, we had growth for the following experimental setups:
- Transformation using direct digested Puc19 from the Mastermix D(MM) digestion approach
- Transformation using digested and ligated Puc19 from the Mastermix digestion approach L(MM)
- Transformation using digested and ligated Puc19 from the No Mastermix digestion approach L(No MM)
- Positive control set up transformed E. coli bacteria with undigested and unligated Puc19 plasmid)
From the results we obtained after transformation as seen above, we had no growth for the following experimental setups:
- Transformation using direct digested Puc19 from the No Mastermix (No MM) digestion approach
- Negative control set up transformed E. coli bacteria with undigested and unligated Puc19 plasmid
This lab experiment we performed gave us the results that we envisioned. The results implied that the Mastermix digestion approach that we used did not ensure that all or some of the plasmids were digested, hence the growth from the direct transformation. We then concluded that using the No Mastermix approach for digestion for future lab experiments would be ideal.
2.0 SECOND LAB EXPERIMENT WITH SYNTHESIZED PROJECT PARTS
We conducted the second lab experiment with the rest of the synthesized parts for our project. We used the 1:10 concentrated plasmid, No Mastermix approach for digestion and made new competent cells for this last experiment with the final set of the synthesized parts.
We used the same reagents and micro volumes from the first lab experiment conducted with synthesized genetic parts for the project.
The results we obtained from the transformations are displayed below.
Figure 9: Results obtained from Digestion and ligation of synthesized IDT and Twist parts (a) Gold inducer – Kill switch module (b) Arsenic inducer – Kill switch module (c) Ferric Inducer module
From the results displayed above, we observed a few colonies but still with low transformation efficiency. However, we took some of these colonies and grew them in the media overnight. We then used this overnight culture to perform a mini prep. After the mini-prep, we performed a Polymerase chain Reaction to amplify the inserted genes for each of the synthesized DNA in the Puc19 plasmid.
2.1 PCR EXPERIMENTFor the PCR, we had two tubes from two colonies of the Fe (I & II), two tubes from two colonies of Au and three tubes from three colonies of the As. We included a Puc19 plasmid DNA as control. Below are tables for PCR conditions and PCR reagent volumes we used.
After the PCR, we run a gel with the amplified DNA parts. Below is a gel image of the gel run on the PCR amplified genetic parts.
Figure 10: Gel electrophoresis results obtained for amplified DNA parts
2.3 CONCLUSION AND TROUBLESHOOTINGThe anticipated band size for the genetic inserts used in this PCR gel are listed below.
From the gel image above, it can be clearly observed that none of our genetic inserts were present (columns 2-8). However, the amplified portion of the puc19 in the PCR, which had approximately 184 base pairs, was present in each of the genetic inserts.
We suspected that one of the reasons the result came out the way it did might be because of the annealing temperature used for the PCR which was very close to the TM of the primers, impeding the amplification of the insert. Other reasons could be that the ligation of the parts might not have worked properly. We think that this is a possibility because the number of colonies was small. Since the plasmid cannot ligate itself we think that maybe few plasmids were not digested and lead to small transfection efficiency.
To trouble shoot if the PCR did not work correctly, we aimed to run another gel with the DNA parts directly from the mini prep. Unfortunately, we ran out of SYBR gold stain, used to visualize the DNA in the gel. We ordered some SYBR gold, but it did not arrive before the deadline to perform the experiment and hence could not troubleshoot this reasoning.
📝 Once we have DNA staining dye, we would change the annealing temperature to increase the difference between the annealing temperature and TM .
📝 We would order new genetic parts for our project and troubleshoot digestion and ligation of the parts with the Puc19 plasmid.
Our solution proposed the deployment of live E. coli cells in the environment, so we ensured biosecurity and safety while maximizing the efficiency of our biosensor through hydrogel encapsulation. During the project's progress, the team moved from theoretical research to experimental lab work to test the theory of hydrogel1 encapsulation. Below is the protocol used and the results obtained. We could not add the hard shell of polyacrylamide hydrogel because our order did not arrive in time. Alginate forms hydrogel in di-cationic solutions (e.g., Ca2+, Ba2+) and has been used for various biomedical applications.
Figure 11: Cell encapsulation in hydrogel capsules (a) schematic of the DEPCOS platform. (b) The process of core shell encapsulation of cells. Droplets of 2.5% alginate with engineered E. coli were crosslinked in a CaCl2 solution to form the soft core of the bead. Outsourced from (Tang, Tham, Liu, & Lu, 2020)
Manufacturing the Alginate CoresA 10% alginate solution was made by dissolving medium viscous alginate in Distilled water, followed by autoclaving at 120 degrees to ensure sterility. A fresh bacterial culture with GFP and Red Mcherry was then mixed with the alginate solution in a one-to-one volume ratio to reach a final alginate concentration of 5 w.t%. This bacteria alginate premix was loaded into a syringe and dropwise added into a beaker of a CaCl2 solution (Fig. 11a) to form a beadlike droplet (Fig. 11b) with controllable diameters ranging from 2.5mm to 3mm. Immersing the droplet in the CaCl2 solution for a few minutes solidified the droplets formed.
Figure 12: (left) The process of core shell encapsulation of cells. 2.5% alginate solution (right) Droplets of engineered E. coli crosslinked in a CaCl2 solution to form the soft core of the hydrogel
Figure 13: Hydrogel containing E coli cells with (a) alginate containing cells with GFP (b) alginate containing cells with Red mcherry (c) Alginate