Improve

Achievements

We improved BBa_J45503, a promoter which is active at low temperatures by removing non-essential parts of the Part sequence given in the registry.
We improved expression at 16 °C 2-fold.
Despite stronger expression, we maintained signal to noise ratio of expression at 16 °C vs 37 °C at 2:1.
We improved the ratio of expression at 16 °C to room temperature 1.5-fold, tightening expression closer to the desired temperature.

About the use of a cold-induced promoter in a project for the warmest climates

Mosquitoes are attracted by body heat1. While all biologic systems produce heat, we did not expect the S.H.I.E.L.D. to produce enough heat to attract mosquitoes based on its small biomass alone. Therefore, we decided to employ BBa_K410000, a registry Part submitted by GeorgiaTech in 2010. BBa_K410000 is composed of cold-shock promoter HybB (BBa_J45503) and alternative oxidase a1 from Nelumbo nucifera (NnAOX1a), which we submitted as basic Part as BBa_K2588018 since it was not individually submitted by GeorgiaTech themselves. NnAOX1a salvages electrons from the respiration chain and converts their potential energy to heat2. Combined in BBa_K410000, they have the function of inducing heat production at low temperatures.

The S.H.I.E.L.D. is heavily focused on sustainability. We liked GeorgiaTech’s approach of coupling heat production to a negative feedback loop to ensure E. coli does not take damage from overexpression of a transembrane protein salvaging electrons from their respiration chain. That HybB is active only at low temperatures, and only shows low expression at higher temperatures, compliments our philosophy of only using moderate expression to ensure viability of cells over long periods of time. We expect the intrinsic feedback loop of BBa_K410000 to work even at higher temperatures, despite its overall lower expression.

BBa_J45503 encodes more than just the HybB promoter

When we analysed BBa_K410000 to confirm that it is properly designed, HybB caught our attention. Despite claiming to be a promoter sequence, an alignment against the E. coli genome revealed that BBa_J45503 not only comprises of the clean promoter sequence, but also contains the Hyb0 5’ UTR and the first 100 bp of Hyb0 coding sequence. When using BBa_J45503 in a standard assembly with a downstream gene of interest, we expect this to result in a conflict in translation of Hyb0 with the gene of interest encoded on the mRNA, which we expect to reduce the expression of the gene of interest for the benefit of truncated Hyb0 as unwanted side-product.

Improvement

We decided to improve HybB by reducing it to its essential promoter sequence. To not rely only on the E. coli genome RefSeq annotation, we employed Berkeley Drosophila Genome Project’s online tool for prokaryotic promoter prediction to predict the promoter sequence and find the most likely transcription start site (Fig. 1)3.

**Fig. 1 Map of BBa_J45503 with annotation based on RefSeq and promoter prediction.**

Since we do not know how temperature induction of HybB promoter works, we decided to keep everything upstream of the predicted promoter sequence. Based on our assumption, that conflicts in translation lead to weakened GOI expression, the upstream sequence would not be detrimental, whatever function it has. For our optimized version of HybB promoter, we removed the sequence downstream of the predicted promoter, keeping the first ten bases of predicted mRNA.

Results

We amplified the trimmed HybB promoter by PCR from E. coli genomic DNA, introduced it into pSB1C3, which we submitted as BBa_K2588001, as well as upstream of BBa_E0240 in pSB1C3, which we submitted as BBa_K2588049. Since BBa_J45503 is not distributed via the registry, we amplified it by PCR from E. coli genomic DNA ourselves, and repeated the cloning process described above. We transformed competent E. coli DH5α with both constructs, and incubated them at 16 °C, room temperature (23 °C), and 37 °C for 12 hours. We measured GFP fluorescence in all samples using a plate reader and normalized it using OD₆₀₀ (Fig. 2).

**Fig. 2 Improvement of BBa_K2588001 over BBa_J45503.** GFP fluorescence of *E. coli* cultures expressing both HybB-Promoter/GFP combinations at different temperatures was measured. Values are mean + SD from three technical replicates. Significances: * corresponds to p<0.05, ** corresponds to p<0.01, *** corresponds to p<0.001.

As expected, we improved GOI expression of HybB promoter by removing Hyb0 5’ UTR and CDS from BBa_J45503. This also includes the background activity at 37 °C, which means that background expression of BBa_K2588001 roughly corresponds to BBa_J45503 expression at 16 °C. However, the ratio of expressions of 16 °C and 37 °C remained stable at 2:1. Intriguingly, the activity patterns of BBa_J45503 and BBa_K2588001 differ in their relative expression level at room temperature. The expression difference of BBa_K2588001 between 16 °C and 25 °C was increased 1.5 fold against BBa_J45503, leading to a tighter expression pattern of our improved part.

Discussion

Our Part improvement led to the expected result of a higher overall expression. While higher expression at low temperatures is a desirable result for many applications at low temperatures, background expression at 37 °C increased too, which might have detrimental effects in applications requiring a low background. Still, BBa_K2588001 maintains the same signal to noise ratio of 2:1 comparing expression at 16 °C and 37 °C. At 25 °C, relative expression in BBa_K2588001 is lower than in BBa_J45503, making it an ideal candidate for applications that require a tighter expression at low temperatures. For these, employing a weaker RBS would decrease expression at all temperatures, only leaving the tightening effect.

Team:Hamburg/Improve