The improvement of function was focused on BioBrick BBa_K2165004 built by iGEM16_Washington, which is a copper-induced promoter in S.cerevisiae. This promoter has been proved to be a good element for Cu ion’s detection with many different eukaryotes. We have also characterized this promoter with different copper concentrations and induction time.

However, this promoter is much longer than other Cu-induced promoter used in prokaryotes, since it adapts to much more complicated conditions in eukaryotes. We redesigned this promoter by deletion of some unrelated bases and kept the core sequence which control the main function of this promoter. After experimental verification, we got the new part (BBa_K2407000).

On the other hand, we found this promoter has a leakage expression and its response range in S. cerevisiae is not as wide as what some previous team described. Under this condition, we used error-prone PCR to reduce the leakage expression and enlarge the response range. Finally we got new parts with less leakage expression and lager response ranges, and they are: (BBa_K2407013), (BBa_K2407014), (BBa_K2407015), (BBa_K2407016).

Functional Improvement

Redesign of CUP1 promoter

Transcription with CUP1 promoter is activated by sequence-specific transcription factors which bind to the CUP1 upstream activating sequence (CUP1 UAS). This sequence contains five binding sites for the Cu2+-dependent transcriptional activator ACE1/CUP2. The CUP1 UAS is necessary and sufficient for rapid and robust activation of this transcription.

First, based on the part (BBa_K2165004) provided by iGEM16_Washington, we read researches about this promoter and ensured the core part consisted of 5 ACE1 binding sites (UAS), 2 TATA boxes, and one initiation element. The complex of ACE1 and copper ions will bind the promoter, which causes the activation of CUP1 promoter with TATA boxes’ help. ACE1 complex’s binding directly increases the possibility for TBP (TATA-Box Binding Protein) to bind the promoter, which can enhance the expression.

Fig. 1. structure of redesigned CUP1 promoter used in our project, based on BBa_K2165004

We redesigned the part sequence provided by iGEM16_Washington. We deleted irrelevant bases on the two ends of this promoter and retained the core sequence. In this way, this promoter played its key role with less bases.

Strains of S. cerevisiae BY4742 containing either BBa_K2165004-yEmRFP and BBa_K2407000-yEmRFP with an initial OD600 of 0.1 were grown for 24 hours in SC-URA medium at 30 degrees Celsius, and then were induced with 0.1 mM Cu2+. Samples were tested with fluorescent microplate reader after 1, 3, 6, 12, and 24 hours.

Fig. 2. structure of redesigned CUP1 promoter used in our project, based on BBa_K2165004

From the result we got. The expression of yEmRFP in yeast with the two promoters were very similar. We could speculate that the core function and sequence hasn’t been damaged during our redesign.

Error-Prone PCR

In our experiment, we noticed that CUP1 promoter still has a certain degree of leakage expression. To make a better biosensor, we planned to reduce the leakage expression and increase the sensitivity. To reach this goal, we took the fluorescence intensity at both induction or not into evaluation indexes.

The technology of error-prone PCR was taken into our experiment. Although there are many methods to introduce genetic diversity into a parent sequence, error-prone PCR is the most common way of creating a combinatorial library based on a single sequence. By adding some heavy metal ions into the PCR buffer and preparing dNTPs with different composition, new mutants were introduced into CUP1 promoter.

Fig. 3. The procedure for error-prone PCR experiment

The library of promoter mutants obtained from error-prone PCR were ligated into plasmid pRS416 with two restriction sites (BamHI and XbaI). After that, we enriched different plasmids from E.coli and established the plasmid library with 132 samples. Then, different plasmids were transferred into S.cerevisiae BY4742 to test the fluorescence intensity under different conditions.

We tested the leakage expression and response range for each ideal mutant we got. Click here to see the result. The sequencing result can be also found in the improved parts’ information: BBa_K2407013 (EP-3), BBa_K2407014 (EP-5), BBa_K2407015 (EP-9), BBa_K2407016 (EP-28).