Overview
In the whole process from the preliminary determination of the project to the successful implementation of the project, we iterated and adjusted the selection and design of the expression system of the degradation module and suicide module in BCAID through consulting the literature, communicating with experts and other iGEM teams. (For more details: Integrated human practice) Among them, the expression system of degradation module was finally changed from the initial anaerobic expression system to LightOn light-controlled expression system, and because of the particularity of the vector we chose, we adjusted the specific design of LightOn to adapt to its vector. After several iterations, the expression system of suicide module was finally decided as REDMAP light-controlled expression system and its system design is adjusted. After Test, both systems were initially proved to be able to operate successfully in our chassis cells.
Engineering--The blue light-activated Bacterial Cellulose(BC) degradation module
Design 1
The application of cellulose scaffold in skin tissue is limited by its non-degradability in human body. We designed an anaerobic induction expression system, aiming to express and secrete cellulase when the wound healed to an appropriate extent, thus degrading the cellulose scaffolds and preventing possible scar formation.
Build 1
The oxygen content decreased during wound healing, while the anaerobic respiration of yeast could cause ethanol fermentation. We considered to conduct cellulase expression system in E. coli and use the anaerobic induced nirB promoter (Fig.1). When the wound healed to a certain extent, the reduction of oxygen volume induced the expression of cellulase (endoglucanase and exocellulase) to achieve scaffold degradation.
Fig.1 Anaerobic conditions induction of cellulase expression
Learn 1
Through searching relevant literatures and discussing with other teams, we concluded that it is inappropriate to use E. coli as the expression host. First of all, E. coli is a conditional pathogen and its co-culture with BC membrane, a wound healing scaffold, has safety threatens. Besides, according to wound healing, it may be difficult to start cellulose membrane degradation artificially. Meanwhile, it is hard to determine the relationship between oxygen concentration and wound healing. Most importantly, the implanted scaffold could also lead to oxygen reduction during cell attachment.
Design 2
In order to make it more flexible to start the expression of cellulase to degrade BC membrane based on wound healing, we intended to introduce a LightOn gene expression system activated by blue light. In this system, transcription factors can be rapidly activated after blue light irradiation, which can start transcription and induce expression of cellulase to degrade cellulose scaffolds. LightOn system consists of two parts: one part is GAVPO, a transcription activator regulated by blue light, and it contains three structural units, namely Gal4, VVD, and p65 activation domain. Another part is the target transcription unit containing target gene regulated by the transcription activating factor, and it includes 5xUASG, TATA box and cellulase gene.
Build 2
We constructed two lentivirus mammalian expression vectors: one vector realized the function of constitutive expression of GAVPO protein, and another used T2A as a linker to realize blue light induction to initiate the expression of Cex(exo-β-1,4-glucanase), CenA(endoglucanase) and Bglx(β-glucosidase) (Fig. 2).
Fig.2 Construction of lentivirus mammalian expression plasmids. a.The plasmid containing GAVPO. b.The plasmid containing three cellulases.
Learn 2
Because of the unusually stable photoactivated state of VVD, the LightOn system is extremely sensitive to light, we can minimize any potential toxicity of blue-light irradiance on cells. 2A peptides, a kind of self-cleaving peptides, can make a transcript produce multiple proteins. Among them, T2A is a high cleaving efficiency peptide. However, lentivirus has only one transfer plasmid encoding the target protein in the packaging process, and the others are packaging plasmid and envelope plasmid. In order to introduce the whole LightOn system, GAVPO and cellulase need to be fused in one plasmid and transferred into cells.
Design 3
However, the combination of GAVPO and three cellulases in one vector may exceed its load resulting in packaging failure or low viral titer. We designed to construct three lentivirus plasmid vectors, and each vector contained transcription factor GAVPO and one of the cellulases. It is anticipated that when the three transformed cell lines are co-cultured with BC membrane, we will realize the final degradation of cellulose into glucose.
Build 3
We decided to construct three lentivirus expression vectors that could be induced to express Cex, CenA and Bglx, respectively (Fig. 3).
Fig.3 Schematic representation of LightOn system components
Learn 3
In the promoter region of the LightOn system, poly A and pause sites can eliminate background interference. However, through discussion with experts, we found that there was a problem in the original sequence. Lentivirus belongs to retrovirus, and poly A cannot be inserted between 5'LTR- Δ U3 and 3'LTR-Δ U3 of lentivirus packaging vector. Otherwise, the transcription would be terminated in advance during packaging, thus leading to packaging failure or drop in virus titer.
Design 4
In order to prevent lentivirus packaging from failing due to the early termination of transcription during packaging, we chose to remove poly A and pause site sequences at the N-terminal of the LightOn promoter region. At the same time, the target transcription unit induced by blue light was switched to the upstream of GAVPO constitutive expression to reduce the background and prevent CMV promoter from affecting the transcription of other downstream genes.
Build 4
We removed poly A and pause site sequence in LightOn promoter region of the three expression vectors, and switched the blue-light induced expression module to the upstream of GAVPO (Fig.4). Moreover, we carefully checked the sequence of GAVPO elements and optimized the base sequence on the basis of codon preference to ensure that GAVPO can be constitutively and efficiently expressed.
Fig.4 Schematic representation of the LightOn system after adjustment
Learn 4
GAVPO was constitutively expressed, and it required blue light irradiation for GAVPO to dimerize and bind to the UASG sites in LightOn promoter region. After GAVPO dimer binding to DNA sequences, it activated the downstream cellulase expression. The signal peptides of eukaryotes can guide the protein output in animal cells. However, the signal peptides in our cellulase genes are prokaryotic and may not play a role in eukaryotic cells.
Design 5
To enable eukaryotic cells successfully express and secrete cellulase, we removed the N-terminal prokaryotic signal peptide of cellulase gene and fused the eukaryotic secreted signal peptide. At the same time, we designed to fuse the protein tag at the C-terminal of the protein, aiming to detect and purify the protein expression.
Build 5
We removed the prokaryotic signal peptide at the N terminal of the gene encoding cellulase, fused the eukaryotic expression signal peptide IL-2 signal sequence.
Test 5
The lentivirus expression vectors pCex, pCenA and pBglx were mixed with the transfection reagent Lipo8000, respectively. And we completed transient transfection of these plasmids to HEK293T cells. After 24 hours, the cells were exposed to blue light for 3 hours. After 48 hours, cells and culture medium were collected. The supernatant of the culture medium and the cell lysis products were analyzed using the SDS-PAGE method. We further analyzed it through Coomassie brilliant blue staining and Western blot experiment.
Fig.5 Western blot analysis of cellulase. Notes: CenA molecular weight is about 48 kDa; Cex molecular weight is about 51 kDa; Bglx molecular weight is about 66 kDa. a. and b. are the Western blot analysis diagram of cell lysates, and c. is the Western blot analysis diagram of culture medium supernatant.
Learn 5
The results of Coomassie brilliant blue showed that both the supernatant and the cell lysate samples had a large content of proteins in the target size of 45 kDa - 60 kDa. Western blot analysis proved that GAVPO and cellulase were successfully expressed in cells after transient transfection, and the exocrine secretion of endoglucanase (CenA) was verified(Fig.5).
Engineering--The red light-activated suicide module
Design 1
Engineered fibroblast ATCC CRL-2522(BJ) will secrete cellulase to degrade bacterial cellulose. After the cellulose scaffold is degraded, BJ completes its own function. To avoid the BJ continuing to secrete cellulase and causing an unnecessary organismal response, we hope to design a suicide switch. Since degradation is regulated by the blue light system, the suicide switch needs to be regulated by another non-conflicting system. When reviewing the literature, we still focused on optogenetics, hoping to achieve the self-apoptosis through another light control system. We first chose to construct PhyB / PIF6 red and far-red light response switch 1, when the BJ is exposed to the red light, photochrome PIF6 will interact with the PhyB component and bring the VP16 transcriptional activation domain close to the promoter, which will recruit the RNA polymerase-associated components to initiate the transcription and expression downstream of MazF（Fig.1）.
Fig.1 PhyB / PIF6 red and far-red light response switch
Build 1
According to the principle of starting the system, we constructed two main basic plasmids, pKM01 and pKM02. The bicistronic expression vector (pKM01) has two cistrons (Fig.2). In the first cistron, the N-terminal fragment of PhyB (amino acids 1–650) is fused to the VP16 herpes simplex virus–derived transactivation domain and to an NLS (from simian virus 40 large T antigen). In the second cistron, the N-terminal 100 amino acids of PIF6 are fused to the tetracycline repressor TetR. Translation of the second cistron is induced by a polioviral internal ribosome entry site IRESPV. The response vector (pKM02) comprises 13 repeats of the TetR-specific operator tetO fused to the minimal human cytomegalovirus immediate-early promoter PhCMVmin, which controls the expression of the reporter, MazF (Fig.3).
Fig.2 The bicistronic expression vector
Fig.3 The response vector
Learn 1
However, after the preliminary design, we learned that the blue light can cause the weak expression of the red light system (Fig.4). This means that when the blue light control system performs the degradation function, a few cells will die at the same time, which will greatly reduce the efficiency or even prevent the degradation of Bacterial Cellulose(BC).
Fig.4 Production of SEAP under different light1
Design 2
In order to avoid the possible interference caused by blue light, we replaced the original system with orthogonal FRL-v2 system2, which will make BJ stimulate bacterial photoreceptor protein BphS to synthesize c-di-GMP under far red light. In the presence of c-di-GMP, FRTA3 (Constituted by the c-di-GMP-binding domain BldD, the transcriptional activation domain p65, and VP64) will form a homodimer, then specifically bind and activate the PFRL-V2 promoter, finally express the downstream toxin protein MazF to initiate suicide (Fig.5).
Fig.5 The Orthogonal FRL-v2 system
Build 2
The plasmid construction needs include three main components (Fig.6): pWS46 (PhCMV-BphS-2A-YhjH-pA), pGY32 (PhCMV-FRTA3-pA; FRTA3, p65-VP64-NLS-BldD), and pXY34(PFRL2.13a-MazF-pA).
Fig.6 Plasmid components a.pWS46 vector b.pGY32 vector c.pXY34 vector
Learn 2
When we finished designing the plasmid map, we discussed with Prof. Liang whether the overall experimental design was reasonable, and he reminded us of some points for attention about lentivirus packaging and infection. Because our engineered fibroblasts need to have both blue light regulated degradation and red light regulated suicide function, each effective cell should be infected by both lentiviruses, respectively. In order to increase the efficiency of the same cell being co-infected by two system lentiviruses, the lentiviruses should be fewer. Therefore, Prof. Liang suggested that we could combine the three plasmids of the FRL-v2 system into one plasmid, and then package only one red light system lentivirus according to the target plasmid, so as to increase the efficiency of secondary infection of the same cell. However, when we decided to try to combine the three plasmids, we found that there is a size limit on the sum of lentiviral plasmid, and we had no way to package all the components of the system in one virus. To solve this problem, we have to look for another newer and smaller red light system again.
Design 3
By reviewing extensive literatures, we finally found a new red / far-red light system REDMAP3 applied in mammals. When exposed to red light (660 nm), the transactivator (FHY1–VP64) can specifically bind to the light sensor domain (ΔPhyA–Gal4) in the presence of the photosensitive pigment PCB, and the combined protein complex translocates into the nucleus where it can bind to its synthetic promoter (P5 × UAS, 5 × UAS-PhCMVmin) to initiate expression of MazF. Following exposure to far-red light (730 nm), the transactivator dissociates from the light sensor domain (ΔPhyA–Gal4), thereby terminating the expression of MazF (Fig.7). Because the photosensitive pigment PCB is a small molecule of chromophore on phycocyanin, which can be directly added externally, the REDMAP system does not need the synthesis and expression of photoreceptor protein.
Fig.7 The new red / far-red light system REDMAP
Build 3
We added the trans-activator (FHY1-VP64), the photosensor domain (ΔPhyA -Gal4), the synthetic promoter (P5 × UAS,5×UAS-PhCMVmin), and the target gene (MazF) to the plasmid core region of the packaging lentivirus (Fig.8a). We also adjusted the plasmid after communicating with another team Peking. To prevent the strong CMV promoter from affecting the transcription of subsequent genes, we regulated MazF to the upstream of the CMV promoter. On this basis, We put different labels to the red light starting element and MazF to facilitate subsequent Western blot inspection (Fig.8b).
Fig.8 REDMAP plasmid a.The original design plasmid b.The adjusted plasmid
Test 3
We verified the function of red light suicide by transient transfection of HEK293T cells, and it was found that almost all cells entered the death state 46 hours after transient transfer.The expression of the intracellular target gene MazF and the red light element could also be seen by the SDS-PAGE and Western blot experiments.
Learn 3
According to the experimental results, the red light system we built can effectively realize the function of biosafety.
Reference
1 Müller, K., Zurbriggen, M. D. & Weber, W. Control of gene expression using a red- and far-red light-responsive bi-stable toggle switch. Nat Protoc 9, 622-632 (2014).
2 Shao, J. et al. Smartphone-controlled optogenetically engineered cells enable semiautomatic glucose homeostasis in diabetic mice. Sci Transl Med 9, (2017).
3 Zhou, Y. et al. A small and highly sensitive red/far-red optogenetic switch for applications in mammals. Nat Biotechnol 40, 262-272 (2022).
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