In Silico Assembly of Genetic Parts Using Benchling

After preliminary research, we identified the important parts needed to create a lithium detector, its pathfinder element, Arsenic, and the genetic parts required for bioleaching. We moved on to designing the plasmid in silico using Benchling. The plasmids we designed contained two assembly standards: the Biobrick and Golden gate assembly methods. All the DNA sequences we designed contained a Biobrick prefix and suffix and a BsaI recognition sequence. When the BsaI restriction enzyme digests the genetic sequence, a 4bp overhang remains at the 5’ end of the DNA sequence, see Figure 1.

Figure 1: DNA sequence skeleton

A successful assembly is influenced by the level of ligation fidelity of the overhangs. Our choice of the overhang base pairs was guided by a paper by Potatov et al [1]. The combination of base pairs we chose for ligation had a ligation fidelity of 99%. Additionally, having dual assembly standards allows us to have the flexibility to switch between multiple assemblies. This design also allows us to troubleshoot our assemblies if experiments do not yield the desired outcome. Furthermore, our selection of the two assembly standards was influenced by the pJUMP24-1A(sfGFP) backbone we used that contained both assembly standards. We used the pJUMP24-1A(sfGFP) because it contains a superfolded GFP that would allow us to screen colonies after assembly and transformation.

Lithium Detector

After preliminary research, we identified the important parts needed to create a lithium detector, its pathfinder element, Arsenic, and the genetic parts required for bioleaching. We moved on to designing the plasmid in silico using Benchling. The plasmids we designed contained two assembly standards: the Biobrick and Golden gate assembly methods. All the DNA sequences we designed contained a Biobrick prefix and suffix and a BsaI recognition sequence. When the BsaI restriction enzyme digests the genetic sequence, a 4bp overhang remains at the 5’ end of the DNA sequence, see Figure 1.

Figure 2: Lithium sensor DNA sequence

The most significant part of our designs was the lithium sensing module. The module contains a BsaI cut site, 4 base pairs, and a bio brick prefix. The promoter initiates the biological circuit. The nhAa-I used for the sensing module was attained from a paper by White et al [2], which outlines the nhAa-I riboswitch could be used to detect the presence of Lithium ions. Once the nhAa-I senses the presence of lithium. The ribosome binding site binds to the ribosome and allows for the translation and expression of the chromoprotein eforRed indicating the presence of Lithium Ions. The terminator is important in stopping the biological circuit.

Arsenic Detector

Figure 3: Arsenic sensor DNA sequence

The 2022 Ashesi_Ghana team identified Arsenic as a pathfinder element for Gold. Balaram & Sawant also identified Arsenic as a pathfinder element for most Lithium containing elements such as Lepidolite and Spodumene [3]. Detecting the presence of Arsenic in a specific place increases the probability of finding lithium and increases the probability of the E. coli bacteria correctly detecting the presence of lithium. The DNA sequence contains an arsenic sensing module that triggers the expression of the fluorescent protein AmilGFP that glows in the presence of blue light to show the presence of Arsenic.

UV kill Switch

Figure 4: UV kill switch DNA sequence

This DNA sequence consists of the two assembly standard sequences, the UV promoter, ribosome, T4 endolysin and terminator. This creates a sequence that triggers the expression of T4 endolysin which causes the bacteria to self-destruct when exposed to UV light. This sequence is essential as it serves as a safety measure to prevent the bacteria from escaping into the environment.

HiPIP & pH Resistance sequence &Tetrathionate Hydrolase

Figure 5: HiPIP & pH resistance

This sequence provides a very significant element to the bioleaching module. It contains the HiPIP with a constitutive promoter, RBS and terminator. This part works together with the pH resistance gene to facilitate the reduction and oxidation of ions during bioleaching and at the same time allow the E. coli bacteria to survive in the harsh bioleaching pHs.

HiPIP & pH Resistance sequence &Tetrathionate Hydrolase

Figure 5: HiPIP & pH resistance

Figure 6: Tetrathionate Hydrolase

Benchling Digestion and Ligation of parts

The following plasmids were successfully digested and ligated in the Benchling software giving us a high probability that the assembly will be successful when carried out in the lab. Unfortunately, this did not prove to be correct for the bioleaching plasmid as it failed several times during synthesis.

Lithium Sensing Module and Arsenic Sensing Module

The sensing modules were assembled using the benchling digestion and ligation assembly tool. This allowed us to select the parts to be assembled, followed by selecting the enzyme to use for cutting the parts. Since the overhangs created after digestion were compatible, benchling demonstrated that the parts are compatible, thereby leading to the assembled plasmid. The parts for the Lithium module were the lithium part, the UV_T4 endolysin and the pJUMP24-1A(sfGFP) backbone. The Arsenic sensor on the other hand contained arsenic part, the UV_T4 endolysin

Bioleaching Plasmid

The assembly of the bioleaching assembly contains the HiPIP, Hp Resistance gene, Tetrathionate Hydrolase and the pJUMP24-1A (sfGFP) backbone plasmid.

Figure 9: Ligated Plasmid with Arsenic Detector and UV Kill Switch

In silico Digestion of pJUMP24-1A(sfGFP)

In order to verify that we were using the correct vector backbone in the reaction, we used benchling to perform an in-silico digestion using the BsaI restriction enzyme. From the digestion, the expected size of the uncut pJUMP24-1A (sfGFP) was 4399bp. As shown the virtual digest, the expected band sizes of the digested plasmid were 3560 bp and 839 bp respectively.

Figure G1: Expected results from Gel electrophoresis on Benchling

Parts Assembly

Once the parts arrived from IDT (October 5th despite being ordered on July 17th) we prepared the stock concentrations of 10ng/µl of the Lithium and Arsenic and ran a golden gate assembly reaction. We then transformed the assembled plasmids (Lithium Sensing module and Arsenic Sensing Module) into three E. coli strands: BL21, NEB-10Beta, and NEB-5alpha using the Golden gate assembly protocol. Inspection of the different plates after incubation was done using the naked eye and under blue light. The results for the homemade competent cells (BL21, NEB10Beta). The results are as follows:

The plates with the BL21 cells had no growth indicating a problem with the cells. However, there was visible growth in the NEB 10-beta cells. A yellow color could be observed with the naked eye, indicating the presence of GFP. Since the plasmid contained a GFP gen, we also viewed the bacterial plates under UV light. The cells were glowing under UV light, indicating that the assembly did not work as the GFP was not knocked out through the assembly.
These are the results using a commercial competent cell NEB 5-alpha.

There was visible cell growth in the plates. The color was observable using the naked eye and under UV light.
Since the assembly was unsuccessful, we took a step back to troubleshoot. Our first troubleshooting step was to check if the BsaI-HFv2 enzyme used was working effectively. We ran a digestion with the old BSA1 and new BsaI-HFv2 enzymes and ran a Gel Electrophoresis.

GEL Electrophoresis

We ran the digested pJUMP24-1A(sfGFP) on a 1% agarose gel and used NEB 1kb DNA ladder (Figure G3) to identify the inserts. The results of the Gel electrophoresis are shown below. Figure G1 shows the expected results from the Benchling simulation, and G2 shows the results from the lab experiment. Comparing G1 and G2, the ladder maker in G2 was between 3560 bp and (850bp), which corresponded to the fragments in G2, meaning the size was as expected, ndicating that Bsal was working correctly. The results showed that the new and old Bsal were cutting and working correctly. The new BsaI-HFv2 produced more intense bands than the old BSa1 enzyme indicating that it was more efficient at digesting DNA.

Golden gate assembly using NEB protocol

We tried another golden gate assembly protocol by NEB to assemble the Lithium and Arsenic plasmids in hopes that the protocol would yield a different outcome. After the end of the experiment, the plates with Arsenic had bacteria colonies that glowed under UV light. However, the plates which contained the Lithium Sensing plasmid had a total of 11 colonies and 5 of them were not glowing under UV light. This indicates the colonies that were not glowing (approx. 50%) had possibly been carrying a successfully assembly. More tests are planned to prove this assumption.

Hydrogel synthesis

Phase One Experiment

This part aimed to encapsulate E. coli bacteria in a Sodium Alginate hydrogel to create a safely contained and sensitive sensing module. The first step was to determine the hydrogel concentrations with the best structural integrity and simultaneously allow for the growth of the bacteria. The experiment carried out was with three difference concentrations of bacteria (1.00, 0.1 OD, and 0.01 OD). These bacterial densities were resuspended in 3mls of different concentrations of Sodium Alginate (2.5%, 5%, and 10%) crosslinked with 5% Calcium Chloride.

Figure R1:2.5%,5% & 10% sodium alginate solution

After placing the hydrogels in a petri dish and incubated at 37 °C. the plates were monitored for 5 days.

Phase One Results

The 10% alginate hydrogels demonstrated the best structural integrity, while the 2.5% hydrogels had issues forming individual entities. Additionally, the OD of the cell culture (cell densities) influenced the extent of visible color changes in the hydrogel after the incubation period, with the OD of 1.0 resulting in the most significant change.

Implications of Phase One Results

The results suggest that a higher cell culture density (1.0 OD) expresses a deeper blue color change in the hydrogels, while lower densities (0.1 and 0.01 OD) do not. We recognized the need for higher cell concentrations to achieve a visible color change in the hydrogels. As a result, we conducted another experiment using cells with an optical density (O.D.) of 1.0 and above. The hydrogels with a 10% alginate concentration displayed the best structural integrity, and the cells within these hydrogels grew optimally. We utilized hydrogels from a 10% alginate concentration to ensure favorable cell growth and viability conditions.

Phase Two Experiments

We proceeded to carry out another experiment with higher optical densities. We used aeblue cells ( E.coli contain colorimetric plasmid) with OD measurements of 4.0, 3.0, 2.0.

Figure R2: 2.0 O.D, 3.0 O.D & 4.0 O.D 10% sodium alginate solutions

Phase Two Results

The color of the hydrogel with the cells with an optical density of 4.0 expressed a deeper blue color from the beginning, followed by the cell culture with an optical density of 3.0 and 2.0. After a day of incubation, the cells presented an even more pronounced blue color. The was no change in color from day two until day 5.

We also noted that the cells' optical density (OD) appears to have a direct relationship with the expression of the blue colour in the hydrogels, which corroborated our earlier findings. The bacteria may have stopped expressing the blue colour due to a lack of nutrients or entering the lag phase of growth.

Phase Three Experiment

From these findings we moved on to using cells between the optical density of 0.1 and 1.0 ODs. For our final experiment we used the Optical densities of 0.25, 0.50, 0.75 and 1.00 OD to find out which concentration would give us a stable cell growth and color change.

Figure R2: 0.25 O.D, 0.5 O.D, 0.75 0.D & 1.0 O.D 10% sodium alginate solutions

Phase Three Results

There was no noticeable color change in the hydrogels made. One assumption was that the cells were too old, so they were no longer expressing the blue color.

Viability Test

Since the hydrogels had not expressed any blue color we ran a viability test on the hydrogels, to check if the cells were still growing and expressing the blue color. Below are the results we obtained, confirming that the cells were alive and growing.

Figure R4: Overnight culture of 0.75 O.D, 0.5 0.D & 0.25 O.D cell solution.

Since the hydrogels had not expressed any blue color we ran a viability test on the hydrogels, to check if the cells were still growing and expressing the blue color. Below are the results we obtained, confirming that the cells were alive and growing.

The hydrogel beads did not turn blue as expected. However blue dots were observed on some of the beads.

After the encapsulation, we assessed cellular viability. Below are the picture of the cells pre-shaking. Cells were grown before being centrifuged and collected for an overnight culture, producing a recognizable blue mass of cells at the bottom of the tube. Viability was assessed through naked-eye observation, optical density measurement, and color assessment of the collected group after centrifugation.

Figure 11: Hydrogels in incushaker.

Figure 11: Hydrogels in incushaker.

Hydrogel Synthesis Using GFP

To further corroborate the results we repeated the experiment using hydrogels containing bacteria with a green florescent protein. This approach should have an increased sensitivity than the colorimetric approach.

Over a three-day observation period, we examined the Petri dishes and recorded measurements daily. Although the color change was less visible to the naked eye, we viewed the plates under UV light, revealing a notable fluorescence of the hydrogel spheres containing the cells.

GFP plates under blue Light

Conclusion

From our results, we successfully identified the optimal concentration required to create a hydrogel with strong structural integrity while simultaneously facilitating optimal bacterial growth. In the context of our project, it was crucial for the bacteria to remain within the hydrogel for a minimum of 5 days when deployed into the ground. To ascertain whether the cells would continue to grow and interact with the Lithium and Arsenic ions during this period, we conducted a viability test spanning 5 days. Our results demonstrated that the cells remained viable throughout this duration. To further validate our findings, we employed two different types of bacteria, namely aeblue and GFP, and successfully substantiated our claims. Although these experiments were not conducted with the inducible cells, one we create the inducible cells and characterize their induction and sensitivity we can directly substitute the required concentration of cells to create the biosensing unit.

[1] V. Potatov, J. Ong, R. Kucera, B. Longhorrst, and K. Billot, “Comprehensive profiling of four base overhang ... - ACS publications,” ACSPublications, https://pubs.acs.org/doi/10.1021/acssynbio.8b00333 (accessed Oct. 11, 2023).
[2] N. White, H. Sadeeshkumar, A. Sun, N. Sudarsan, and R. R. Breaker, “Lithium-sensing riboswitch classes regulate expression of bacterial cation transporter genes,” Scientific Reports, vol. 12, no. 1, 2022. doi:10.1038/s41598-022-20695-6
[3] V. Balaram and S. S. Sawant, “Indicator Minerals, Pathfinder elements, and portable analytical instruments in Mineral Exploration Studies,” MDPI, https://www.mdpi.com/2075-163X/12/4/394#:~:text=In%20the%20case%20of%20lithium,and%20spodumene%20(Table%201). (accessed Oct. 12, 2023).