Part contributions

In this project we mainly created four new composite parts for the part library of iGEM. Only only did we document their basic biological information but also provide the original experimental data, which develop the Part Registry of iGEM innovation.

Table 1. Part contributions overview

Part number	Part name	Contribution type
BBa_K484000 2	pCMV-3Tag-1A-OGN	New part
BBa_K48400 08	RNAi-Ready pSIREN-RetroQ-ShRNA-1	New part
BBa_K484000 9	RNAi-Ready pSIREN-RetroQ-ShRNA-2	New part
BBa_K48400 10	RNAi-Ready pSIREN-RetroQ-ShRNA-N	New part

① Add a new Composite Part: BBa_K4840002 (pCMV-3Tag-1A-OGN)

1)Construction of OGN overexpressed plasmid

In this work, we first constructed OGN overexpressed plasmid. In brief, DNA fragment of OGN was amplified by using the cDNA of ATDC5 cells and then inserted into the pCMV-3Tag-1A vector. The E. coli strains solution transformed with constructed plasmid were spread onto LB solid medium plate containing corresponding antibiotic and cultured at 37℃ overnight (Figure 1).

Figure 1. Intiatives utilized by Lambert iGEM to increase accessibility to diagnosis for coronary artery disease (CAD).

We randomly selected several monoclonal colonies from the plate for cultivation, and extracted plasmid DNA from the cultured bacterial solution. The oligo DNA in plasmid was identified by using agarose gel electrophoresis (Figure 2A). Our results show that a band of about 5000 bp is present in sample 2 and 8, suggested that pCMV-3Tag-1A-OGN plasmid were constructed successfully. At the same time, we sent the positive monoclonal antibody to the company for sequencing. Figure 2B showed that the gene sequence was correct and there was no mutation. It proves that pCMV-3Tag-1A-OGN plasmid was successfully constructed.

Figure 2. Electrophoretic gel (left) and Sequencing (right) Verification of pCMV-3Tag-1A-OGN plasmid

2)Identification of ITS-induced ATDC5 cells

In order to explore the function of OGN on the matrix metabolism of chondrocytes. We firstly added ITS to the growth medium to induce the differentiation of ATDC5 cells. Since mature chondrocytes contain a large amount of proteoglycan, which can be stained blue by Alcian Blue, we performed related assays in ITS-induced ATDC5 cells. As shown in Figure 3, our cultured ATDC5 cells have differentiated into chondrocytes.

Figure 3. Identification of chondrocytes by microscope

3)The increase of OGN expression improved matrix metabolism in IL-1β-treated chondrocytes

Next, we established an in vitro research model of OA by using IL-1β as stimulator on chondrocytes and detected the mRNA and protein levels of OGN in the cells by using qRT-PCR and western blotting. As shown in the Figure 4, the mRNA and protein levels of OGN in IL-1β-stimulated chondrocytes were significantly decreased compared with those of control group, that is consistent with the clinical observation that OGN expression in chondrocytes from osteoarthritis patients is reduced. Moreover, both the mRNA and protein levels of OGN in chondrocytes transfected with previously constructed pCMV-3Tag-1A-OGN plasmid were notably increased compared to the mock group, further indicating the overexpressed plasmid was successfully constructed.

Figure 4. pCMV-3Tag-1A-OGN plasmid antagonisted IL-1β-induced inhibition on OGN

expression in chondrocytes (A) qRT-PCR results; (B) western blot results

Additionally, we investigated whether OGN participates in the pathogenesis of OA. As shown in Figure 5, mRNA levels of matrix metabolism-related genes such as Aggrecan, Col-2 and Sox9 were downregulated, while MMP3 and MMP13 mRNA levels were upregulated in IL-1β-induced chondrocytes. These results indicated the disruption of matrix metabolism in chondrocytes. Notably, OGN overexpression antagonized such IL-1β-induced effects in chondrocytes. Thus, all results suggests that OGN is a promising therapeutic target for OA.

Figure 5. OGN overexpression antagonisted IL-1β-induced matrix metabolism disorder in chondrocytes

②Add 3 new Composite Parts: BBa_K4840008 ~BBa_K4840010

(RNAi-Ready pSIREN-RetroQ-ShRNA-1、RNAi-Ready

pSIREN-RetroQ-ShRNA-2、RNAi-Ready pSIREN-RetroQ-ShRNA-N)

1)Construction of OGN gene silencing plasmids

Moreover, we designed and synthesized two interference sequences of OGN gene for the construction of OGN gene silencing plasmids and performed similar experiments to explore whether the OGN is involved in the initiation and progression of OA.

Briefly, the forward and reverse sequences of ShRNA were connected by annealing. The obtained DNA fragments was inserted into the RNAi-Ready pSIREN-RetroQ vector. The E. coli strains solution transformed with constructed plasmid were spread onto LB solid medium plate containing corresponding antibiotic and cultured at 37℃ overnight (Figure 6).

Figure 6. Monoclonal colonies of E. coli transformed with OGN gene silencing plasmids (A) RNAi-Ready pSIREN-RetroQ-ShRNA-1; (B) RNAi-Ready pSIREN-RetroQ-ShRNA-2; (C) RNAi-Ready pSIREN-RetroQ-ShRNA-N

We randomly selected several monoclonal colonies from the plate for cultivation, and extracted plasmid DNA from the cultured bacterial solution. The oligo DNA in plasmid was identified by using agarose gel electrophoresis (Figure 7A). At the same time, we sent the positive monoclonal antibody to the company for sequencing. Figure 7 B. C.D showed that the gene sequence was correct and there was no mutation.

It proves that RNAi-Ready pSIREN-RetroQ-ShRNA-1, RNAi-Ready pSIREN-RetroQ-ShRNA-2, RNAi-Ready pSIREN-RetroQ-ShRNA-N plasmid were successfully constructed.

Figure 7. Electrophoretic gel (A) and Sequencing (B.C.D) Verification of RNAi-Ready pSIREN-RetroQ plasmid

2)OGN gene silencing can induce matrix metabolism disorder in chondrocytes

As shown in Figure 8 and Figure 9, the mRNA and protein levels of OGN were significantly decreased in IL-1βgroup and OGN-Sh-1, 2 group compared with those of Control group or OGN-Sh-N group. In addition, the results of qRT-PCR revealed that the decrease of OGN expression can induce the disruption of matrix metabolism in chondrocytes, indicative of the regulatory role of OGN in OA.

Figure 8. RNAi-Ready pSIREN-RetroQ-ShRNA-1 and 2 plasmids inhibited the expression of OGN in chondrocytes (A) qRT-PCR results; (B) western blot results

Figure 9. The decrease of OGN expression induced matrix metabolism disorder in chondrocytes

Other Contributions

Due to the complex pathological mechanism, high incidence and high disability rate, OA brings huge medical care burden and economic burden to the whole society. However, there is no clear and effective drug to improve the condition like rheumatoid arthritis in the treatment of OA. The completion of this research project is not only expected to provide new drug targets for the treatment of OA, but also provide new ideas for the development of OA drugs. And it can provide theoretical data support for other iGEM.

The future iGEM team can be further studied. You find a vector carrying OGN into chondrocytes with targeting and high efficiency, and use this vector to carry OGN plasmid to overexpress OGN in chondrocytes. Such carriers can refer to viral carriers and nanomaterial carriers ;You construct an OA animal model to observe whether overexpression of OGN can delay the process of OA in vivo. It is necessary to observe the biological safety of overexpression of OGN in vivo, including the detection of whether there is damage to important organs.