Team:XMU-China/Contribution
Since XMU-China is on the Open Track this year, BOTH of the Contribution (Bronze Criterion #5) and Validated Contribution (Silver Criterion #1) are recorded on the SAME page below.
For the Contribution (Bronze Criterion #5), we completed the experimental characterization of the previous parts (BBa_K118022 and BBa_K2564000) and added the data of them to the corresponding BioBricks.
For the Validated Contribution (Silver Criterion #1), We have designed 37 new BioBricks for our project, and all of these parts have been submitted to the Registry. We experimentally validated that most of the new BioBricks work as expected.
All of these may be helpful to other teams. We hope it will make some contribution to the iGEM community.
We’ve summarized some achievements related to experimental characterization about existing Parts from the Registry of Standard Biological Parts. And all of the data have been added to the corresponding BioBricks.
| BioBricks | Codes in the lab | Quantitative Characterization | |
|---|---|---|---|
| Contribution | BBa_K2564000 | bgl1A | HPLC of the enzymatic activity |
| BBa_K118022 | cex | MUC Test of the enzymatic activity |
1) cex (BBa_K118022)
T7-RBS (BBa_K525998) was used for protein expression. And crude enzyme solutions were used for characterization. Methylumbelliferyl cellobioside (MUC) was degraded into methylumbelliferone and cellobiose in the presence of exoglucanase. Fluorescence, emitting from methylumbelliferone at a wavelength (λ=366 nm) of ultraviolet light, was used to determine the activity of Exoglucanase.
Fig. 1. Assay for cex activity determination by using MUC. (A) Supernatant and control. (B) Broken supernatant and control.
The results showed that the T7-RBS-cex expression lead to enhancement of fluorescence intensity, which verified the enzyme activity of exoglucanase. It further demonstrated that the Exoglucanase encoded by T7-RBS-cex was able to degrade methylumbelliferyl cellobioside into methylumbelliferone and cellobiose.
2) bgl1A (BBa_K2564000)
T7-RBS (BBa_K525998) was used for protein expression. And crude enzyme solutions were used for characterization. Both substrate (cellobiose) and products (glucose) are reducing sugars in the reaction in which Beta-D-glucosidase that encoded by bgl1A works as catalyst. However, the traditional methods for the determination of reducing sugars are not suitable. Thus, high performance liquid chromatography based on ion-regulated partition chromatography was employed for characterization here.
Fig. 2. Standard Working Curve of HPLC. (A) Retention time of different concentrations of cellobiose and glucose standards. (B) Standard Working Curve of cellobiose and glucose. (C)&(D) Chromatogram of non-target peaks from LB liquid and PBS buffer.
As the experiment progressed, cellobiose gradually decreased to zero and glucose increased. This indicated that Beta-D-glucosidase degraded cellobiose into glucose, which verified the enzyme activity in broken supernatant of T7-RBS-bgl1A. (The medium supernatant didn’t have enzyme activity).
Fig. 3. The results of HPLC. (A) supernatant of T7-RBS-bgl1A. (B) broken supernatant of T7-RBS-bgl1A.
37 new BioBricks were designed for our project, all of which have been submitted to the Registry. A complete documentation has been provided here, in which BioBricks were new designed by ourselves and in working order.
(A) Cooperative Part
In this part, two kinds of secretion systems were constructed (YebF signal protein and Kil secretion cassette) to secrete our target proteins (Bgl1A, CenA and Cex) this year.
We validated the secretion of related protein first, and then characterized the enzyme activity. Most of the parts worked well to perform their normal functions. All data related are recorded here.
| BioBricks | Codes in the lab | Characterization | ||||
|---|---|---|---|---|---|---|
| SDS-PAGE of secretion | Enzyme activity | |||||
| Extracellular | Extracellular | Extracellular | Extracellular | |||
| Validated Contribution of Cooperative Part |
BBa_K2922000 | YebF-bgl1A | √ | √ | X | √ |
| BBa_K2922001 | YebF-cenA | √ | √ | √ | √ | |
| BBa_K2922002 | YebF-cex | √ | √ | √ | √ | |
| BBa_K2922006 | T7-yebF-bgl1A | √ | √ | X | √ | |
| BBa_K2922007 | T7-yebF-cenA | √ | √ | √ | √ | |
| BBa_K2922008 | T7-yebF-cex | √ | √ | √ | √ | |
| BBa_K2922012 | 109-kil-bgl1A | √ | √ | X | √ | |
| BBa_K2922013 | 112-kil-bgl1A | √ | √ | X | √ | |
| BBa_K2922014 | 114-kil-bgl1A | √ | √ | X | √ | |
| BBa_K2922015 | 109-kil-cenA | √ | √ | √ | √ | |
| BBa_K2922016 | 112-kil-cenA | √ | √ | √ | √ | |
| BBa_K2922017 | 114-kil-cenA | √ | √ | √ | √ | |
| BBa_K2922018 | 109-kil-cex | √ | √ | √ | √ | |
| BBa_K2922019 | 112-kil-cex | √ | √ | √ | √ | |
| BBa_K2922020 | 114-kil-cex | X | √ | √ | √ | |
a) SDS-PAGE of secretion
1) YebF series
Parts related:
| BioBricks | Codes in the lab | |
|---|---|---|
| Validated Contribution of Cooperative Part |
BBa_K2922000 | YebF-bgl1A |
| BBa_K2922001 | YebF-cenA | |
| BBa_K2922002 | YebF-cex | |
| BBa_K2922006 | T7-yebF-bgl1A | |
| BBa_K2922007 | T7-yebF-cenA | |
| BBa_K2922008 | T7-yebF-cex |
Result: All of the target genes were expressed and secreted out into the medium, which meant that this secretion system worked well.
Fig. 4. SDS-PAGE by Sliver staining. (A) genetic circuit of yebF-bgl1A; (B) genetic circuit of yebF-CenA; (C) genetic circuit of yebF-cex; (D) yebF-bgl1A(BBa_K2922006), target bands: 63.4 kDa; (E) yebF-CenA(BBa_K2922007), target bands: 57.5 kDa; (F) yebF-cex(BBa_K2922008), target bands: 62.0 kDa.
2) kil secretion cassette series
Parts related:
| BioBricks | Codes in the lab | |
|---|---|---|
| Validated Contribution of Cooperative Part |
BBa_K2922012 | J23109-kil-bgl1A |
| BBa_K2922013 | J23112-kil-bgl1A | |
| BBa_K2922014 | J23114-kil-bgl1A | |
| BBa_K2922015 | J23109-kil-cenA | |
| BBa_K2922016 | J23112-kil-cenA | |
| BBa_K2922017 | J23114-kil-cenA | |
| BBa_K2922018 | J23109-kil-cex | |
| BBa_K2922019 | J23112-kil-cex | |
| BBa_K2922020 | J23114-kil-cex |
Result: Except the BBa_K2922020, it comes to the conclusion most of the target genes were expressed and secreted out into the medium, which means most of the secretion systems work well.
Fig. 5. SDS-PAGE by Coomassie blue staining. (A) genetic circuit of 109-kil-bgl1A; (B) genetic circuit of 112-kil-bgl1A; (C) genetic circuit of 114-kil-bgl1A; (D) J23109-kil-PT7-RBS-bgl1A(BBa_K2922014), target bands: 53 kDa. (E) J23112-kil-PT7-RBS-bgl1A(BBa_K2922012), target bands: 53 kDa. (F) J23114-kil-PT7-RBS-bgl1A(BBa_K2922013), target bands: 53 kDa.
Fig. 6. SDS-PAGE by Sliver staining. (A genetic circuit of 109-kil-CenA; (B) genetic circuit of 112-kil-CenA; (C) genetic circuit of 114-kil-CenA; (D) J23109-kil-PT7-RBS-cenA(BBa_K2922017), target bands: 47 kDa. (E) J23112-kil-PT7-RBS-cenA(BBa_K2922015), target bands: 47 kDa. (F) J23114-kil-PT7-RBS-cenA(BBa_K2922016), target bands: 47 kDa.
Fig. 7. Results of SDS-PAGE. (A) genetic circuit of 109-kil-cex; (B) genetic circuit of 112-kil-cex; (C) genetic circuit of 114-kil-cex; (D) J23109-kil-PT7-RBS-cex(BBa_K2922020, Sliver staining), target bands: 47 kDa. (E) J23112-kil-PT7-RBS-cex(BBa_K2922018, Coomassie blue staining), target bands: 47 kDa. (F) J23114-kil-PT7-RBS-cex(BBa_K2922019, Coomassie blue staining), no target band.
b) Enzyme activity
1) Beta-glucosidase(Bgl1A)
Parts related:
| BioBricks | Codes in the lab | |
|---|---|---|
| Validated Contribution of Cooperative Part |
BBa_K2922012 | J23109-kil-bgl1A |
| BBa_K2922013 | J23112-kil-bgl1A | |
| BBa_K2922014 | J23114-kil-bgl1A |
Method:HPLC based on ion-regulated partition chromatography.
Result:Broken supernatant but not culture supernatant of all the target genes had enzyme activity. It meant that Beta-glucosidase coded by this gene worked well, which could degrade the cellobiose into glucose.
Fig. 8. The results of HPLC. (A) genetic circuit of yebF-bgl1A; (B) genetic circuit of 109-kil-bgl1A; (C) genetic circuit of 112-kil-bgl1A; (D) genetic circuit of 114-kil-bgl1A; (E) yebF-bgl1A supernatant; (F) yebF-bgl1A broken supernatant; (G) 109-kil-bgl1A supernatant; (H) 109-kil-bgl1A broken supernatant; (I) 112-kil-bgl1A supernatant; (J) 112-kil-bgl1A broken supernatant; (K) 114-kil-bgl1A supernatant; (L) 114-kil-bgl1A broken supernatant.
2) Endoglucanase (CenA)
Parts related:
| BioBricks | Codes in the lab | |
|---|---|---|
| Validated Contribution of Cooperative Part |
BBa_K2922015 | J23109-kil-cenA |
| BBa_K2922016 | J23112-kil-cenA | |
| BBa_K2922017 | J23114-kil-cenA |
Method:Congo Red assay.
Result: Both supernatant and broken supernatant of the target gene have enzyme activity. It meant that Endoglucanase coded by this gene worked well, which could degrade long chain polysaccharides into short chains. The former will bind to Congo Red while the latter will be washed off, and then left transparent stain.
Fig. 9. The results of Congo Red assay. (A) supernatant of PT7-cenA; (B) broken supernatant of PT7-cenA; (C) supernatant and broken supernatant of J23109-kil-cenA; (D) supernatant and broken supernatant of J23112-kil-cenA; (E) supernatant and broken supernatant of J23114-kil-cenA.
3) Exoglucanase (Cex)
Parts related:
| BioBricks | Codes in the lab | |
|---|---|---|
| Validated Contribution of Cooperative Part |
BBa_K2922018 | J23109-kil-cex |
| BBa_K2922019 | J23112-kil-cex | |
| BBa_K2922020 | J23114-kil-cex |
Method:MUC Test.
Result:Both supernatant and broken supernatant of the target gene have enzyme activity. It meant that Exoglucanase coded by this gene worked well, which could degrade Methylumbelliferyl cellobioside (MUC) into methylumbelliferone and cellobiose.
Fig. 10. Result of MUC Test.
(B) Aggressive Part
In this part, two kinds of secretion systems were used (NKil secretion cassette and EKil secretion cassette) to secret our target proteins (Colicin-N and Colicin-E1) this year.
First the secretion of related protein was validated. Then the virulence and the effect that secretion ability imposed on the virulence were characterized. Most of the parts worked well to perform their normal functions. All data related are recorded here.
| BioBricks | Codes in the lab | Characterization | |||
|---|---|---|---|---|---|
| SDS-PAGE | Enzyme activity | ||||
| Intracellular | Virulence | Promote by secretion | |||
| Validated Contribution of Aggressive Part |
BBa_K2922024 | Colicin-E1 | √ | √ | |
| BBa_K2922025 | Eimm | √ | |||
| BBa_K2922026 | Ekil | √ | |||
| BBa_K2922027 | Colicin-N | √ | √ | ||
| BBa_K2922028 | Nimm | √ | |||
| BBa_K2922029 | Nkil | √ | |||
| BBa_K2922030 | TEimm | √ | |||
| BBa_K2922031 | TEkil | √ | |||
| BBa_K2922032 | TNimm | √ | |||
| BBa_K2922033 | TNkil | √ | |||
| BBa_K2922034 | TN | √ | √ | ||
| BBa_K2922035 | PN | √ | √ | ||
| BBa_K2922036 | TE | √ | √ | ||
| BBa_K2922037 | PE | √ | √ | ||
a) SDS-PAGE of secretion
1) Imm and kil series
Parts related:
| BioBricks | Codes in the lab | |
|---|---|---|
| Validated Contribution of Aggressive Part |
BBa_K2922025 | Eimm |
| BBa_K2922026 | Ekil | |
| BBa_K2922028 | Nkil | |
| BBa_K2922029 | Nimm | |
| BBa_K2922030 | TEimm | |
| BBa_K2922031 | TEkil | |
| BBa_K2922032 | TNimm | |
| BBa_K2922033 | TNkil |
Result:All of the target genes were expressed successfully, which meant that the related genes worked well.
Fig. 11. SDS-PAGE by Coomassie blue staining. (A) Eimm, target bands: 13 kDa; (B) Ekil, target bands: 4.8 kDa; (C) Nkil, target bands: 5.6 kDa; (D) Nimm, target bands: 15 kDa.
b) Enzyme activity
1) Virulence of Colicin-N and Colicin-E1
Parts related:
| BioBricks | Codes in the lab | |
|---|---|---|
| Validated Contribution of Aggressive Part |
BBa_K2922034 | TN |
| BBa_K2922035 | PN | |
| BBa_K2922036 | TE | |
| BBa_K2922037 | PE |
Method: Inhibition zone experiment by Oxford cup (The sample is obtained from bacteria culture medium)
Result:The sizes of inhibition zone in the experimental group were larger than that in control group, verified that the virulence of Colicin-N and Colicin-N was strong enough to kill other bacteria nearby.
Fig. 12. Inhibition zone experiment by Oxford cup. Experimental group in the left and control group in the right of every petri dish. (A) T7-RBS-Colicin-N (BBa_K2922034). (B) pBAD-RBS-Colicin-N (BBa_K2922035). (C) T7-RBS-Colicin-E1 (BBa_K2922036). (D) pBAD-RBS-Colicin-N (BBa_K2922037).
2) The secretion of Colicin-N and Colicin-E1 to promote virulence.
Parts related:
| BioBricks | Codes in the lab | |
|---|---|---|
| Validated Contribution of Aggressive Part |
BBa_K2922034 | TN |
| BBa_K2922036 | TE |
Method:Inhibition zone experiment by Oxford cup (The sample is obtained from bacteria culture medium).
Results: The sizes of inhibition zone in experimental group were larger than that in control group, which verified that both of secretion of Colicin-N and Colicin-E1 could enhance the effect of inhibition.
Fig. 13. Inhibition zone experiment by Oxford cup. Experimental group in the left and control group in the right of every petri dish. (A) T7-RBS-Colicin-N (BBa_K2922034). (B) T7-RBS-Colicin-E1 (BBa_K2922036).