Part design

Almost all the parts are designed for expressing the needed proteins in our project. We referred to many papers, got many expressing pathways, compared them with each other, and eventually chose 12 pathways. According to our demands, some of the pathways are redesigned, including replacing original parts with some specific parts to regulate the expression of the pathways.

All the parts can be divided into four groups: 2×2 transfer switch, flavor factors, functional proteins, and oxygen-controlled suicide switch. For each of the groups, we composed the basic parts into composite parts and regarded every composite part as an independent module to test and verify its function. All the composite parts have been tested and the results have been shown on the experiment page.

Basic parts

Best basic part

We proudly present our best new basic part: BBa_K4256133 (FNR). FNR is a bifunctional protein that acts both as a hypoxic sensor and a hypoxia-responsive transcription factor. In E. coli and Salmonella, the Fumarate and Nitrate Reduction regulator (FNR) regulates the global response to the transition between aerobic and anaerobic growth. Acquisition of a [4Fe-4S] cluster initiates the formation of FNR homodimers that bind DNA site-specifically and regulate transcription from target promoters. This regulation is environmentally friendly and the two conditions are clearly delineated, allowing for easy treatment of escaped bacteria and ensuring biosafety.

All the basic parts

Composite parts

Best composite parts

Our best composite parts are BBa_K4256666 and BBa_K4256672. They are the bistable switches controlled by both blue light and temperature. The former switch combines a photosensitive, blue light-controlled switch (BBa_K2469004)with two RBSs corresponding to Temperature（25°C） (BBa_K3247005) binding at both sides of the switch. When the temperature rises to 25 ℃ or higher, the RC site (RNase E cleavage site) of the RBS will be truncated by RNase E, which will stop the expression of the downstream gene. At low temperatures(≤25°C), the ARC site (Anti RNase E cleavage site) matches with the RC site complementarily and forms a stem ring structure, which can not be cut by ribonuclease. Thus, the target gene downstream of the Ptrc promoter can express. The latter switch combines a photosensitive, blue light-controlled switch (BBa_K2469004 )with two RBS corresponding to Temperature（37°C） (BBa_K3247005)binding at the end of the switch. When the temperature reaches 37°C or higher, in the absence of light, the structural Helmholtz potential of RBS is at its peak and LacILOV represses the expression of genes under the Prtc-2 promoter, meanwhile allowing the downstream gene of cl: LVA promoter to express. Upon blue light excitation, this repression is lifted, and cI is expressed, able to repress the BBa_R0051 promoter. If the temperature is below 37°C, the target gene downstream of the Ptrc promoter can express. These constructs demonstrate new configuration of a 2*2 bistable switch which allows future teams to use as a reference.

All the composite parts

Part Collection

This part collection represents three kinds of part-design ideas, including multi-regulated, cascade, and bistable pathways. BBa_K4256198 is an oxygen-sensitive suicide switch based on the ccdA-ccdB toxin-antitoxin system that kills engineered bacteria escaping from anaerobic to normoxic environments. Some of the oxygen-sensitive promoters and transcriptional regulatory factors’ genes are also used to regulate this pathway in multiple methods. BBa_K4256011 is a fusion protein’s gene pathway that encodes four enzymes that transform accessible carbon sources such as glucose and glycerol into L-tyrosine. This is a typical cascade pathway and also one of our expression pathways. The cascade reaction is the most efficient for the expression system for which we have confirmed the conditions and that’s the reason we wildly use it. BBa_K4256666 is a bistable switch controlled by both blue light and temperature. This switch combines a photosensitive, blue light-controlled switch with two RBS corresponding to temperature binding at the end of the switch so it can be regulated by natural light and temperature, which serves as a user-friendly regulating method. The bistable pathway makes sure of the efficient and accurate shift between different conditions. These parts are the creations based on a flash of light followed by detailed verifications, so we have high expectations for them.

Improvement

The 2×2 transfer switch is derived from a bistable switch which functions by expressing two different genes in the presence and absence of blue light. We modified it to form two genetic circuits that work exclusively at high and low temperatures respectively. Together, these two circuits make up a transfer switch which intakes a combination of light and temperature and outputs an expected substance out of four options.

This idea comes from orthogonal biology, where we think about controlling as many results as possible with the least number of conditions. We found the light-controlled and temperature-controlled switches, which were separately verified by the previous team. On the basis of considering the orthogonality, we combined these two organically and added some regulatory elements to form a bistable regulation mechanism, and finally made the bistable switches shown in Fig. 1 and Fig. 2.