The p70 fragment with overhang sequence of ALR (Fig 2) was synthesized using PCR with Pfu polymerase and primers ALR 21 and ALR 34. The annealing temperature was 57.9oC and the extension time was 1 min.

Fig2. Introduction of overhangs bases on the p70 lactate sensitive promoter by PCR.

The expected size of p70-Alanine Racemase (ALR) overhang sequence is 196 base pairs (bp). Lanes 1 marks DNA ladder. Lanes 4 and 6, which are replicates of the same reaction, are DNA product of the expected PCR size of 70-ALR overhang band.

PCR amplification of ALR gene (Fig 3) was performed with Q5 polymerase and ALR 35 (forward primer) and ALR 24 (Reverse primer)

Stitching of p70 promoter to ALR was performed by PCR overlapped with Q5 polymerase and ALR 21 (forward primer) and ALR 24 (Reverse primer) as illustrated in the general cloning strategy (Fig 1).

1. PCR overlap to synthesize construct 1 using Q5 polymerase, along with primers ALR 21 and ALR 38
2. Gel extraction, construct 1 and plasmid vector, pSB1C3 were digested by restriction enzymes (XbaI and PstI-HF) for 3 hours at 37 oC, followed by heat inactivation at 80oC for 20 minutes.
3. PCR clean up for construct 1 and obtained 17.9 ng/𝜇l.
4. Gel extraction for the cut vector and obtained 13.8 ng/𝜇l.
5. Insert and vector were ligated overnight at 16oC. The ratio of insert:vector was 3:1.
6. The ligation mix (5 𝜇l) was used to transform 50 𝜇l of competent bacteria (E. coli DH5).

Upon characterization, it appeared that the promoters with strong ribosomal binding site (RBS, BBa_B0034) exhibited high leakiness. Therefore, we designed this series of constructs by replacing the strong RBS by the medium (BBa_B0032: 30% strength of B0034) and weak RBS (BBa_B0033: 1% strength of B0034). Each promoter fragment p70, p62 and J23100 (without RBS) were isolated and amplified by PCR from the construct 1, 2 and 3 respectively. Each promoter fragment (without RBS) was ligated to the ALR-Terminator fragment by PCR overlap using primers having the overhang sequence of either medium or weak RBS.

Fig 9. Synthesis of constructs 4-9.

• Construct 4: p70-32-ALR-Terminator
• Construct 5: p62-32-ALR-Terminator
• Construct 6: J23100-32-ALR-Terminator
• Construct 7: p70-33-ALR-Terminator
• Construct 8: p62-33-ALR-Terminator
• Construct 9: J23100-33-ALR-Terminator

1. PCR amplification of construct 4, 5 and 8. Their expected size is 1404 base pairs (bp). Lane 1 marks the DNA ladder. Lane 2-5, 6-8 and 10-13, which are replicates of the 3 reactions, show PCR products of construct 4, 5 and 8
2. PCR amplification of construct 6, 7 and 9. The expected size of construct 7 is 1404 bps. The expected size of construct 6 and 9 is 1768 bps. Lane 1 marks the DNA ladder. Lane 2-5, 6-8 and 10-13, which are replicates of the 3 reactions, show PCR products of construct 6, 7 and 9.

We performed the characterization process same as what we did with RIOT sensors 1-3 in milestone 6.

• RIOT sensors 4-6 did not show much improvement compared to sensors 1-3. They are also leaky and hence, not suitable to be integrated into the whole system.
• RIOT sensors 7-9 showed encouraging results since they were more sensitive to small changes in lactate concentration.

Culture mammalian cell lines HeLa and HepG2

1. Revive cells from frozen vials and subculturing 80% confluent
2. Enumerate cell density by a hemocytometer. Seed 106 cells into each well.
3. Resuspend with phenol free DMEM and incubate at 37 oC for 6 hours, then remove media and wash with 1X PBS.
4. Add 500 μl of new media (DMEM which is free of phenol and serum) and incubate at at 37oC for 18 hours.
5. Use a pipette to remove the cell supernatant separately and centrifuge at 250 rcf for 3 min to remove cells that may have left in the supernatant.
6. Cell-free supernatant is used in GFP microscop.

GFP Microscopy

1. Set up an overnight culture of bacteria in a culture tube, with 5mL of LB, plus the appropriate antibiotics. Incubate in a shaking incubator at 37oC overnight.
2. Measure the OD600 of the resuspended bacteria culture. Aliquot appropriate volume so that starting OD for each tube is 0.4, shake in the 37oC incubator for approximately 30 minutes.
3. Set up five fresh and labelled 1.5μl microcentrifuge tubes. To each tube, add 20μl of each lactate concentration (0 M, 10-3 M, 10-2 M) and 180μl of the diluted bacteria sample to make up 200μl of reaction mix. To the remaining two tubes, add 40μl of each cell supernatant (Hela and HepG2) and 10μl of the diluted bacteria sample to make 160μl of reaction mix, with 5x dilution of the samples.
4. Incubate in a shaking incubator at 37oC incubator for 3 to 4 hours.
5. Add 5μl of each sample onto the appropriately labelled polylysine slide and cover with the slip and capture images using NIS microscope.

We found that different lactate concentration in the media or HeLa/HepG2’s supernatant induce different level of GFP expression. RIOT sensor 10 appeared to be the most sensitive sensor to lactate.