Parts – TUDA iGEM 2021

Parts

Listed below are the most important basic and composite parts that we designed for our project. Due to lack of time in the lab we were not able to test all parts. Therefore, we only submitted basic parts and one composite part to the registry. All parts we used in the lab have been synthesized by IDT or Twist.

Basic Parts

Table 1. Most relevant Basic Parts that were implemented in our designs.

Part	Type	Description	Length (bp)
BBa_K3985000	Promoter	Promoter P_PA1897 with a QscR-binding site.	300
BBa_K3985013	Promoter and operator	Operator sites O_R1-O_R3 containing the promoters P_R and P_RM of the lambda phage.	80
BBa_K3985021	Promoter	Bacillus subtilis degQ promoter	433
BBa_K3985001	Coding Sequence	LasR, a quorum sensing transcripion factor which binds 3OC₁₂-HSL, codon-optimized for Bacillus subtilis.	720
BBa_K3985003	Coding Sequence	QscR, a quorum sensing transcripion factor which binds 3OC₁₂-HSL, codon-optimized for Bacillus subtilis.	714
BBa_K3985005	Coding Sequence	eGFP, a basic green fluorescent protein, codon-optimized for Bacillus subtilis.	720
BBa_K3985007	Coding Sequence	mKATE2, a basic red fluorescent protein, codon-optimized for Escherichia coli.	699
BBa_K3985009	Coding Sequence	cI, a transcriptional repressor that keeps the lambda phage in lysogenic state.	714
BBa_K3985010	Coding Sequence	TetR, a repressor which regulates the expression of the tetracycline resistance gene.	651
BBa_K3985012	Coding Sequence	RecA730, constitutive active mutant of RecA, a DNA-dependent ATPase.	1062
BBa_K3985016	Coding Sequence	Cre recombinase, Cre-Lox recombination via tetramer forming complex, lox recognition sites	1032
BBa_K3985017	Coding Sequence	mKATE2, a basic red fluorescent protein codon-optimized for Bacillus subtilis with LVA-ssrA degradation tag	744
BBa_K3985018	Coding Sequence	sfGFP, a green fluorescent protein codon-optimized for Bacillus subtilis with a LVA-ssrA degradation tag	765
BBa_K3985024	Coding Sequence	rpsB, ribosomal protein S2 of Bacillus subtilis, required for translation and cell viability	741
BBa_K3985025	Recombination Site	lox66, right element (RE) loxP mutant, recognition sequence for Cre recombinase	34

Composite Parts

We have created several Composite Parts throughout our project. However, since we have had limited time in the laboratory we were able to characterize only one Composite Part. Nevertheless, this was a great success. The genetic circuit for controlled phage induction worked as expected.

Table 2. Composite Parts we characterized this year.

Part	Type	Description	Length (bp)
BBa_K3985026	Composite	Controlled lytic cycle induction utilizing a cI-RecA730 based genetic switch	3742

Pathogen Sensing

In order to detect the acyl homoserine lactone (AHL) 3OC₁₂-HSL, this genetic construct (Figure 1) is designed to express eGFP in presence of AHL molecules. The signaling molecules AHL first bind to QscR which is expressed constitutively through P_veg. After the complex has formed it binds to P_PA1897 and thus, initiates expression of eGFP. Therefore, fluorescence output indicates presence of the AHL. This molecule is a quorum sensing autoinducer originating from P. aeruginosa. Thus, this construct can be used to detect the presence of the pathogen.

Figure 1. Composite part 1 for pathogen sensing. This part includes P_veg, a consensus RBS, qscR, P_PA1897, egfp and the rrnB terminator. The part was expressed in the pDGB3_omega_ 1 vector.

An alternative for the construct above was designed to engineer our genetic switch regarding the DBTL cycle. This alternative consists of lasR instead of qscR and its corresponding promoter P_Lux instead of P_PA1897 (Figure 2).

Figure 2. Composite part 2 for pathogen sensing. This part includes P_veg, a consensus RBS, lasR, P_Lux , egfp and the rrnB terminator. The part was introduced into the pDGB3_omega_ 1 vector.

In addition to these two constructs, each of them was designed with mKATE2 instead of the transcription factor. This enables further characterization of the constitutive and regulated promoters.

Bacteriophage Induction

We designed and tested a genetic circuit for controlled lambda phage induction after a specific input (BBa_K3985026). An overview of the regulatory elements in the genetic circuit can be found in Figure 3. The construct contains cI, the lambda repressor, which keeps the phage in the lysogenic state. Addition of the specific input (IPTG) activates the T7 polymerase and thus the T7 promoter. Therefore, a strain containing the T7 polymerase, like BL21(DE3), is necessary. The activation of the T7 promoter results in the expression of recA730 and tetR, which both act as countermeasures to cI. The produced TetR binds to the P_tetO promoter and herewith prevents the production of cI. RecA730 degrades the remaining cI, whereby the promoter of the lytic cycle in the genome of the lambda phage is no longer repressed and cro is expressed, thus leading to the activation of the lytic cycle.

To be able to represent the activation of the lytic cycle of the lambda phage by a reporter gene (egfp), the operator sequence of the native lambda switch is placed upstream of the reporter gene. The P_RM promoter activates in its native appearance the lambda repressor, while P_R activates the production of Cro and through a signalling cascade, the lytic cycle.

Figure 3. Our genetic circuit for controlled phage induction. This part contains the native lambda switch, including the operator site O_R and the promoters P_R and P_RM, which control the production of eGFP. The expression of egfp is repressed by cI, which is under control of the P_tetO promoter. This promoter is repressed by TetR. The genes tetR and recA730 are under the control of the IPTG inducible T7 promoter. The part was introduced into the pDGB3_alpha_1 vector.

Kill-Switch

We have developed a synthetic genetic circuit that represents a kill-switch for Bacillus subtilis. It is a bidirectional promoter cassette containing P_veg as constitutive and P_degQ as QS-inducible promoter (Figure 4). Another synthetic genetic circuit for a kill-switch proof of concept as invertible promoter cassette with the IPTG-inducible T7 promoter was also designed. All promoters are flanked by the mutant lox sites lox66 and lox71. Cre-Lox inversion of the promoter cassette should be indicated by the transition from sfGFP to mKATE2 fluorescence.

Upon Cre-Lox recombination the constitutive expression is switched from sfGFP to mKATE2 due to the inversion of the promoter cassette. lox71 thus turns to lox72 double mutant having a much lower affinity for Cre recombinase.

Figure 4. Schematic overview of B. subtilis kill-switch cassette design. It includes the most relevant basic parts lox66, degQ promoter (P_degQ), mKATE2 and sfGFP with degradation tag as well as the rrnB terminator.

Registry Parts

Table 3. Used parts from the iGEM parts registry.

Part	Type	Description	Length (bp)
BBa_J23119	Promoter	TetR-repressible constitutive promoter	56
BBa_K823003	Promoter	P_veg, strong constitutive promoter of Bacillus subtillis	237
BBa_R0062	Promoter	P_Lux promoter, activated by the LasR-AHL-complex	55
BBa_J64997	Promoter	T7 promoter, strong promoter for protein production	19
BBa_K090505	Consensus RBS	Bacillus subtilis consensus RBS	11
BBa_K731721	Terminator	T7 terminator from bacteriophage T7	48
BBa_K823029	Coding Sequence	mKATE2 a basic red fluorescent protein codon-optimized for Bacillus subtilis	702
BBa_K886000	Recombination site	lox71, left element (LE) loxP mutant, recognition sequence for Cre recombinase	34

Team:TU Darmstadt/parts