Week One

Digestion and Isolation of pSB1C3 Backbone.

The purpose of digesting the pSB1C3/PArsRGFP construct was to separate the backbone from the former GFP insert. Tacoma RAINmakers sought to isolate the pSB1C3 backbone employed in their 2017 construct, as the GFP reporter complex was no longer desired. Apparent disadvantages of GFP indication in biosensors are ultraviolet readings. The RAINmakers prefered a chromoprotein that produces color in the visible spectrum. Enzymes XbaI and SpeI were used to cleave the terminator sites of the vector, freeing the pSB1C3 backbone. NEB resources confirmed that the Cutsmart Buffer 2.1 is the most compatible with these particular enzymes, providing an optimized environment for digestion. Combining the reagents listed in Table 1.0, the reaction was set at 37ºC in a water bath for 1 hour and 45 minutes. This reaction was completed in duplicate to increase statistical probability of desired backbone DNA.

Following completion of pSB1C3 digestion, Tacoma RAINmakers employed a standard procedure listed in the protocol page as “Agarose Gel Electrophoresis.” The purpose of this gel was to confirm if the backbone DNA had been successfully isolated from the undesired GFP insert. As cited in Figure 1.1, the expected pSB1C3 bands were located at 2000bp. A gel extraction process, also outlined in the protocol section, was completed in order to remove and contain the digested pSB1C3 DNA.

Week 2

Digestion and Ligation of spisPink, amilCP, and PcArsR Inserts

Using IDT stock solutions of chromoprotein and arsenic regulatory DNA, Tacoma RAINmakers completed a standard restriction digest (see protocol page for further information). The notable difference between insert and backbone digestion are the enzymes. Each insert included a SalI enzyme site, which is not compatible with the BioBrick suffix/prefix of the vector. Instead, the SalI site is used to ligate the chromoprotein to PcArsR, rendering the ends of this complete insert as compatible for sticky-end ligation to pSB1C3. After combining reagents listed in Table 2.0, reaction was held in a water bath at 37°C overnight.

Once the SalI site in spisPink, amilCP, and PcArsR was successfully stick-ended, Tacoma RAINmakers were prepared for ligation of each chromoprotein to the arsenic regulator. 60ng of each part were used in order to ensure that there would enough DNA material for the ligation. Having combined the substances from Table 2.1, the reaction was set to ligate overnight at 16°C. Afterwards, SalI was heat inactivated at 80°C, ensuring a complete denature, since the enzyme was no longer required.

Week 3

Digestion and Ligation of Insert (amilCP/spisPink + PcArsR) to pSB1C3 Backbone

Following the ligation of the chromoprotein and PcArsR, the complete insert was digested with enzymes compatible with our pSB1C3 backbone. This process allows sticky-ended ligation in the next step, which increases the chance of a proper insert-backbone ligation. 84 ng of insert DNA was pipetted into the reaction alongside the other reagents mentioned in Table 3.0. The digestion was set at 37°C in a water bath for 1 hour and 30 minutes.

Tacoma RAINmakers combined the reagents listed in Table 3.1 to ligate the completed insert to the vector. The reaction included a negative control that contained only vector DNA. A notable process involved in ligation reactions is calculating DNA volumes. Typically, a 1:3 ratio of vector to insert ensures that there is a balance of both parts. The RAINmakers employed the NEBioCalculator to determine how many moles were in 1ng of vector (2070bp) and 1ng of insert (1488bp). This calculation translated to 0.8µL of vector and 20µL of insert. The ligation occurred at 16ºC overnight and was heat inactivated at 80ºC for 20 minutes the following morning.

Initiation of Individual Chromoprotein and Regulator Plasmid Design

A standard procedure in the Tacoma RAINmakers project is PCR amplification. This process is listed under the protocol page as “Insert PCR Amplification.” In preparation for ligation of inserts (amilCP, spisPink, and PcArsR) into the vector, all insert DNA must be amplified from its original limited stock. Once the PCR reaction has exited the thermocycler, gel electrophoresis must be employed to assess the efficacy of the amplification. As pictured in Figure 3.2, both the spisPink and amilCP bands successfully appeared at about 1000bp, and the PcArsR expressed at about 550bp. Unfortunately, the PcArsR negative control produced DNA bands, which suggested contamination during the PCR amplification process.

Following a successful PCR amplification confirmed by the gel, Tacoma RAINmakers performed a standard gel extraction. With much more insert DNA, RAINmakers were prepared to design three additional plasmids containing single inserts for isolated testing.

Step 4

Use RNADuplex to calculate binding energies between all remaining mutant ASDs with wildtype SD.

The library is narrowed down again by discarding those candidates with a binding energy less than -1 kcal/mol with the wild type SD sequences. This prevents the orthogonal ribosomes developed from the candidate ASDs from binding with wild type SD sequences over orthogonal mRNA, which ensures orthogonality of the engineered ribosomes. Details

Step 5

Narrow library by eliminating mutant ASD/Wildtype SD pairs with binding energy <-1.0 kcal/mol.

The library is narrowed down again by discarding those candidates with a binding energy less than -1 kcal/mol with the wild type SD sequences. This prevents the orthogonal ribosomes developed from the candidate ASDs from binding with wild type SD sequences over orthogonal mRNA, which ensures orthogonality of the engineered ribosomes. Details

Figure S3. Ensure orthogonality of chosen sequences.

Step 6

Use RNAFold to estimate secondary structure formation of 16s rRNAs containing mutant ASD sequences.

We use RNAfold from the ViennaRNA package to calculate the secondary structure for the full 16s rRNA. Details

Step 7

Narrow library by eliminating sequences that lead to 16s rRNA having secondary structure formed at the ASD region.

Those candidates with secondary structure in the ASD regions are discarded, as this would impair their ability to carry out translation. Details

Figure S4. Elimination of sequences that lead to secondary structure complications.

Step 8

Obtain all translation initiation regions (TIRs) from the chosen bacteria's genome.

Next, we obtain all the translation initiation regions (TIRs) from the genome. Details

Figure S5. Overview: Obtaining all translation initiation regions from the Coding Region.

Figure S6. Extracting the TIRs in different situations.

Step 9

Use RNADuplex^[3] to calculate binding energy of each remaining mutant ASD with those TIRs.

We use these TIRs in conjunction with RNAduplex once again to calculate the binding energies of the remaining candidate ASDs with those TIRs. Details

Step 10

Rank candidate mutant ASDs based on number of strong interactions between the ASD sequence and the TIRs from the bacteria genome.

Candidates are then ranked based on their binding energies with the host TIRs. Candidates with higher binding energies (which are less likely to bind with host TIRs) are given preference, since they are more likely to remain orthogonal to the host processes. Details

Reference

[1] Darlington, A.P.S., Kim, J., Jiménez, J.I., & Bates, D.G. (2018). Dynamic allocation of orthogonal ribosomes facilitates uncoupling of co-expressed genes. Nature Communications, 9, 695.

[2] Ding, Y., Chan, C. Y., & Lawrence, C. E. (2005). RNA secondary structure prediction by centroids in a Boltzmann weighted ensemble. RNA, 11(8), 1157–1166. http://doi.org/10.1261/rna.2500605

[3]“TBI - RNAduplex - Manpage.” Accessed September 12, 2018. https://www.tbi.univie.ac.at/RNA/RNAduplex.1.html.