Team:XMU-China/Results
What work we have done:
Assayed the enzymatic activities of three cellulases separately.
Verified that YebF could be secreted out of the cell.
Verified that YebF-cellulase fusion protein could be secreted out of cell.
Assayed the enzymatic activity of YebF-cellulase fusion protein out of and inside cell.
Verified that cellulases could be secreted out of cell with the help of Kil secretion cassette.
Assayed the enzymatic activity of cellulases out of and inside cell in the presence of Kil secretion cassette.
Verified that cellobiose could be degraded by beta-glucosidase intracellularly.
Work has not finished yet:
Quantitative analysis for the enzymatic activity of CenA.
Get more information of our experiments, please click here.
The Expression of Cellulases
In order to degrade cellulose, we selected three genes from different sources: bgl1A (BBa_K2564000), cex (BBa_K118022) and cenA (BBa_K118023). Cex and CenA degraded cellulose into cellobiose, and Bgl1A degraded cellobiose into glucose (1). We use T7 promoter and RBS (BBa_K525998) to express these proteins at a high level.
The constructed plasmid was transformed into E. coli BL21 (DE3). Positive colonies that were selected by chloramphenicol preliminarily and then by colony PCR, while finally confirmed by sequencing were cultivated and induced by IPTG to express cellulases. The supernatant of culture, namely sup, was obtained by centrifugation. And the total protein was gained by ultrasonication. The lysate underwent centrifugation and its supernatant, namely broken sup, was electrophoresed on a sodium dodecyl sulfate (SDS)-12% (wt/vol) polyacrylamide gel, followed by Coomassie blue staining (Fig. 1-3).
Fig. 1. Gene circuit involved in protein expression and SDS-PAGE analysis of CenA by Coomassie blue staining. (A) Gene circuit was constructed by BBa_K525998 and BBa_K118023 to express endoglucanase (CenA). (B) Target bands of CenA (red arrow, 47 kDa).
Fig. 2. Gene circuit involved in protein expression and SDS-PAGE analysis of Cex by Coomassie blue staining. (A) Gene circuit was constructed by BBa_K525998 and BBa_K118022 to express exoglucanase (Cex). (B) Target bands of Cex (red arrow, 47 kDa).
Fig. 3. Gene circuit involved in protein expression and SDS-PAGE analysis of Bgl1A by Coomassie blue staining. (A) Gene circuit was constructed by BBa_K525998 and BBa_K2564000 to express beta-glucosidase (Bgl1A). (B) Target bands of Bgl1A (red arrow, 53 kDa).
As showed by SDS-PAGE of CenA/Cex/Bgl1A, the target protein can be observed at 47 kDa/47 kDa/53 kDa on the broken sup lanes, but not in the control groups. The results showed that the three cellulases were successfully expressed in E. coli BL21 (DE3). And there were no target protein bands in the supernatant of the culture, indicating that these proteins could not be secreted by themselves.
The Secretion of YebF
YebF (BBa_K1659003) is an E. coli motor protein. When the coding sequences of other protein are fused to the yebF sequence, YebF can help secrete these proteins out of the E. coli membrane (2). YebF was verified to be secreted out of E. coli BL21 (DE3) through SDS-PAGE (Fig. 4B).
Fig. 4. Gene circuit involved in protein expression and SDS-PAGE analysis of YebF by Coomassie blue staining. (A) Gene circuit was constructed by BBa_K525998 and BBa_K1659003 to express YebF. (B) Target bands of YebF (red arrow, 13 kDa).
Target protein from the supernatant of culture was observed at about 13 kDa on the SDS-PAGE gel, showing that YebF was successfully expressed and secreted into the medium.
The Secretion of YebF-cellulase Fusion Protein
In order to secrete our cellulases out of the E. coli membrane, we fused the cellulase gene fragments with yebF gene fragment at the N-terminal using a flexible GS linker (GGGGS) by overlap extension polymerase chain reaction (OE-PCR). PCR products were purified by agarose gel electrophoresis (Fig. 5).
Fig. 5. Agarose gel electrophoresis of OE-PCR product. Lane M: Marker. (A) The target bands of yebF (about 363 bp) and bgl1A (about 1400 bp) in the first step of OE-PCR to fuse the two sequences. (B) The target band of yebF-bgl1A (about 1764 bp) in the second step of OE-PCR. (C) The target bands of yebF (about 363 bp) and cenA (about 1353 bp) in the first step of OE-PCR to fuse the two sequences. (D) The target band of yebF-cenA (about 1731 bp) in the second step of OE-PCR. (E) The target bands of yebF (about 363 bp) and cex (about 1469 bp) in the first step of OE-PCR to fuse the two sequences. (F) The target band of yebF-cex (about 1839 bp) in the second step of OE-PCR. All target bands are showed in red boxes.
These parts were inserted into the expression vectors with T7 and RBS (BBa_K525998) by restriction sites EcoRI and PstI. Then the ligation mixture was transformed into E. coli DH5α, and the correct recombinant one was confirmed by chloramphenicol, colony PCR and sequencing.
The supernatant of culture and cells was separated by centrifugation. The harvested cells was then resuspended, treated by ultrasonication and subsequently centrifuged to gain supernatant, followed by SDS-PAGE and silver staining or Coomassie blue staining (Fig. 6, fig. S1 and fig. S2).
Fig. 6. Gene circuit involved in protein expression and SDS-PAGE analysis of YebF-Bgl1A by silver staining. (A) Gene circuit was constructed (BBa_K2922006) to express YebF-Bgl1A. (B) Target bands of YebF-Bgl1A (red and blue arrows, 63.4 kDa). The character “R” stands for the flexible linker.
The target bands existing in sup (red arrow in Fig. 6B) and broken sup (blue arrow in Fig. 6B) of experimental groups only indicated that YebF-Bgl1A was expressed and secreted out of the cell successfully. The same experiments to YebF-CenA and YebF-Cex also showed similar results that the target bands were observed in sup of experimental groups while not in the control groups (fig. S1-S2). In contrast to cellulases without YebF fused at the N-terminal, which showed no target bands in the sup through SDS-PAGE, and given all of this, we safely concluded that protein YebF contributed to the secretion of these cellulases. In addition, the results of SDS-PAGE confirmed the success of OE-PCR again, while indirectly.
The Characterization of Kil Secretion Cassette
The second secretory mechanism adopted involved the Kil secretion cassette. The kil gene (BBa_K1350001) encodes a kind of lysis protein. Get more information about the secretory mechanism, please click here. However, the secretion can be achieved without cell lysis and death only if the expression intensity of the promoter is proper. Here we applied various constitutive promoters to regulate the expression of Kil protein to investigate its secretion ability. The constitutive promoters we used were BBa_J23114, BBa_J23109 and BBa_J23112 (in the order of strong to weak), respectively. The secretion of Bgl1A was proved by performing SDS-PAGE (Fig. 7). Similar results of CenA and Cex were also obtained except for Cex (114-kil-) (fig. S3-S8).
Fig. 7. Gene circuits involved in protein expression and SDS-PAGE analysis of Bgl1A (114/109/112-kil-) by Coomassie blue staining. (A-C) Gene circuits were constructed (BBa_K2922012, BBa_K2922013 and BBa_K2922014) to express Bgl1A. (E-F) The target bands on SDS-PAGE gels (red and blue arrows, 53 kDa).
Just like the results about YebF, the target bands only existed in both sup and broken sup of experimental groups but not in the control groups. These results confirmed that the Kil secretion cassette successfully helped secrete the target protein out of the cell under the regulation of constitutive promoters. In fact, the light shade of bands in sup convinced us that the bacteria were not dead on a large scale, which further indicated the initiation intensity of J23114, J23109 and J23112 was appropriate enough to avoid cell lysis and death during the culturing process.
However, for the secretory section, no matter the YebF or Kil or some others, a vital issue could not be ignored and skipped was the extracellular enzymatic activity of those target proteins after secretion was achieved successfully. How to choose a most appropriate secretory mechanism so that cellulases can be secreted out of the cell with high activity was the next problem we needed to handle. Actually, we did series of assays for the enzymatic activity of three kinds of cellulases we selected and then made decisions.
Enzymatic Activity of Endoglucanase (CenA)
Congo Red assay was utilized to qualitatively test the enzymatic activity of CenA in the form of crude enzyme, and this method was from iGEM18-UESTC-China, who had a nice collaboration with us this year. As Congo Red only binds to long chain polysaccharides but not short chain, the short chain therefore are washed off during staining procedure resulting in halo formation (3). The results are shown in Fig. 8-9.
Fig. 8. Congo Red assay for enzymatic activity of CenA. Left: sup of PT7-RBS-cenA and blank control (LB liquid medium); Right: broken sup of PT7-RBS-cenA and blank control (PBS).
Areas with the broken sup of CenA added showed due to the hydrolysis of CMC (carboxymethyl cellulose) whereas the blank control didn't show any clearance zones. The obvious difference showed that the broken sup of CenA had enzymatic activity, but the sup of culture had not. This was to say that the enzyme, Endoglucanase (CenA), which was expressed successfully, had a certain level of enzymatic activity to hydrolyze cellulose. Besides, what the results showed was in accordance with the results of SDS-PAGE (Fig. 6B) as well.
The same methods were applied to test the enzymatic activity of the broken sup of YebF-CenA fusion protein and CenA (114/109/112-kil-) and sup of those. The results are shown as follows:
Fig. 9. Congo Red assay for enzymatic activity of YebF-CenA and CenA (109/112/114-kil-). (A) The sup of YebF-CenA and blank control (LB liquid medium). (B) The broken sup of YebF-CenA and blank control (PBS). (C-E) The results of BBa_K2922021, BBa_K2922016 and BBa_K2922017 for CenA (114/109/112-kil-) respectively.
All the areas with CenA crude enzyme, no matter YebF-CenA or CenA assembled with Kil secretion cassette under the regulation of those three promoters added showed clearance zones produced by hydrolysis of CMC (Fig. 9A-9E). While the blank control didn't show any zones of clearance. The results showed that both intracellular and extracellular CenA possessed considerable enzymatic activity.
In order to quantitatively analyze the enzymatic activity of series of CenA, we firstly tried to apply high performance liquid chromatography (HPLC) to separate the products after the substrates were digested by CenA, but the results were not satisfactory enough. Due to the limitation of time, the quantitative analysis for the enzymatic activity of CenA was not finished finally, which was a great regret for us indeed.
Enzymatic Activity of Exoglucanase (Cex)
In order to test the enzymatic activity of Cex, 4-Methylumbelliferyl β-D-cellobioside (MUC) assay was carried out. MUC is broken down into methylumbelliferone (4-MU) and cellobiose in the presence of exoglucanase. The fluorescence intensity of methylumbelliferone can be measured at the emission wavelength of 460 nm (excitation wavelength: 364 nm) (4). In other words, the activity of exoglucanase can be reflected by the fluorescence intensity with time. In this part of assay, all BioBricks containing cex in our project this year were analyzed carefully where the enzymatic activity of sup and broken sup were evaluated respectively (Fig. 10).
Fig. 10. Quantitative analysis for the enzymatic activity of Exoglucanase (Cex) through MUC assay. (A) The fluorescence intensity at different time points of the sup in different experimental groups and the control group. (B) The fluorescence intensity at different time points of the broken sup in different experimental groups and the control group.
The results of sup showed that the supernatant of the culture of control (BBa_K525998) had no enzymatic activity, as there was no change in fluorescence intensity, which was caused by the lack of secretory ability. By contrast to the control, the other experimental groups all presented a certain degree of enzymatic activity, which was reflected by the increase of fluorescence intensity due to the produce of fluorescent 4-MU. It was worth noting that the increase of fluorescence intensity of Cex (109-kil-) group encoded by BBa_K2922018 was most evident and the intensity produced by this group’s sup at 120 min (17037.33) was almost 3 times (even more) higher than that of the other Cex groups (Fig. 10A). Different to sup, the broken sup of all experimental groups showed not so obvious difference among groups (Fig. 10B). Before 90 minutes, the signal of broken sup of Cex (109-kil-) was highest, which convinced us that J23109 was the most proper promoter could be applied in subsequent experiments including demonstration, together with the results of sup.
Additionally, the results of Cex (114-kil-) showed that the enzyme in the supernatant of the culture indeed had activity like the Cex (109-kil-) and Cex (112-kil-) whose secretion had been confirmed through SDS-PAGE, which could be evidence of the secretion of Cex (114-kil-) to some extent.
Quantitative Analysis for the Enzymatic Activity of Beta-glucosidase (Bgl1A)
Given the fact that the beta-glucosidase (Bgl1A) can catalyze the hydrolysis of cellobiose to glucose, we came out that the products (glucose) and substrates (cellobiose) in the reaction system could be separated by high performance liquid chromatography (HPLC) and quantified through the peak areas in the chromatogram, which contributed to analyzing the enzymatic activity of Bgl1A. First of all, we investigated whether the cellobiose and glucose could be separated in our HPLC equipment. Fortunately, these two vital molecules could be distinguished obviously with 5 mM H2SO4 as the mobile phase (retention time of cellobiose: 9.5 min, retention time of glucose: 11.5 min, Fig. 11A). Then, the standard working curves (SWC) of cellobiose and glucose in certain chromatographic conditions were determined and plotted (Fig. 11B), which was the quantitative basis for the latter analysis.
We have noticed that the buffer and medium we used a lot might bring unnecessary affects to subsequent analysis, therefore the LB medium and PBS was tested before we carried out the assay, respectively. The chromatograms of LB liquid medium and PBS presented strong peaks at similar time about 8 min (Fig. 11C & 11D), which would not hinder the analysis of cellobiose and glucose.
Fig. 11. Quantitative analysis was supported by HPLC. (A) The chromatograms of cellobiose and glucose of different concentrations. (B) The SWC of cellobiose and glucose. (C) The chromatogram of LB liquid medium. (D) The chromatogram of PBS.
The quenched reaction systems of different reaction times were analyzed through HPLC (Fig. 12, fig. S9). For the Bgl1A (PT7-RBS-bgl1A), the sup of the culture showed no enzymatic activity as the concentration (conversed according to SWC) of cellobiose and glucose in the supernatant of the culture hardly changed, which was same to that of control group (Fig. 12A). While the broken sup of experimental and control groups showed significant difference. The concentration of cellobiose decreased fiercely with time and the concentration of glucose increased rapidly with time, for the experimental group. While, there was no change of the concentration of the two molecules for the control group. Given all of this, we could summarized that the crude enzyme of Bgl1A (PT7-RBS-bgl1A), just as the results of broken sup showed, had enzymatic activity of a certain level whereas the supernatant of the culture had not. It is worth mentioning that this conclusion can be derived from the comparison of results between all experimental groups and their correspondent control groups in this assay (fig. S9). Regardless of this, the further calculations of the average consuming rate of cellobiose and producing rate of glucose in 0.5 h were done to compare the enzymatic activity of Bgl1A in different experimental groups (Tab. S1). The results of the three constitutive promoters were at a same level, where the consuming rate of cellobiose and producing rate of glucose of J23114-RBS-kil-PT7-RBS-bgl1A was the highest (8.724 g·L-1·h-1 and 8.683 g·L-1·h-1 respectively) among all groups. The enzymatic activity of YebF-Bgl1A was at the lowest level, which may be caused by the fusion of two protein molecules with the steric effect. But it was just our guess without further investigation.
Fig. 12. Quantitative analysis for the enzymatic activity of Bgl1A (PT7-RBS-bgl1A) was supported through HPLC. (A) The concentration of cellobiose and glucose of sup at different times. (B) The concentration of cellobiose and glucose of broken sup at different times.
All experimental groups except Bgl1A (PT7-RBS-bgl1A) showed no enzymatic activity, which confused us much, because the enzyme was actually secreted out of the cell (verified through SDS-PAGE). That is to say that the beta-glucosidase (Bgl1A) cannot function as normal intracellularly when it is secreted out of the cell. In fact, once we had suspected that the conditions caused by our treatment to the samples of sup made the loss or decease of the enzymatic activity of Bgl1A, especially the pH condition of the culture. However, the following experiments we carried out to explore the influence of pH for the enzymatic activity of Bgl1A in the supernatant of the culture was failed (data not shown). In that case, we started to consider whether some changes in the structure of Bgl1A had emerged or not. Unluckily, due to the limit of time, we haven’t continue to investigate this issue further.
Beta-glucosidase (Bgl1A) can Function Intracelluarly
With the help of Prof. Zeng, we knew that the Bgl1A could catalyze the hydrolysis of cellobiose intracellularly in E.coli BL21 (DE3) due to the function of beta-galactoside permease encoded by lacY gene (5). Thus, we carried out the following experiment to prove it. The two recombinant bacteria, one carrying PT7-RBS-bgl1A and the other carrying BBa_K525998 as control, were cultured in different M9 restriction medium with the cellobiose and glucose as the sole carbon source respectively. The growth curves of all these experimental and control groups were monitored through the OD600 value. The results proved that the Bgl1A could function successfully intracellularly, since the bacteria carrying PT7-RBS-bgl1A had grown normally in the restriction medium with cellobiose as the sole carbon source rather than the bacteria of control group (Fig. 13).
Fig. 13. The growth curves for the verification of beta-glucosidase functioning intracellularly.
Brief Summary of Cooperative
Combining the experiments’ results above, the cellulases working effectively and the YebF and Kil secretion cassette functioning successfully was verified anyway. In view of all of this, we finally completed the design and construction of the ultimate circuits which would be applied to the demonstration.
What work we’ve done:
Verified Nimm, Eimm, Nkil and Ekil can be expressed successfully.
Verified the virulence of Colicin-N be able to kill other bacteria nearby successfully.
Verified the virulence of Colicin-E1 be able to kill other bacteria nearby successfully.
Verified the activity of Nkil to promote the secretion of Colicin-N.
Verified the activity of Ekil to promote the secretion of Colicin-E1.
Work hasn’t finished yet:
Characterization of the virulence of Colicin-N and Colicin-E1.
Get more information of our experiments, please click here.
Expression of Nimm、Eimm、Nkil、Ekil
Plasmids pSB1C3 carrying three different BioBricks BBa_K2922032 (T7 promoter-RBS-cni (Nimm)), BBa_K2922030 (T7 promoter-RBS-imm (Eimm)), BBa_K2922033 (T7 promoter-RBS-cnl (Nkil)) and BBa_K2922031 (T7 promoter-RBS-kil (Ekil)) were built and transformed into E.coli BL21 (DE3) successfully.
After culture and induction, the proteins (Nimm, Eimm, Nkil and Ekil) expressed were shown in the graphs of SDS-PAGE gels (Fig. 14).
Fig. 14. Coomassie stain of SDS-PAGE gels. (A) The target bands of Eimm (white box, 13 kDa). (B) The target bands of Ekil (red box, 4.8 kDa). (C) The target bands of Nkil (black box, 5.6 kDa). (D) Nimm (red box, 15 kDa).
The virulence of Colicin-N and Colicin-E1 to kill other bacteria nearby
Plasmids pSB1C3 carrying three different BioBricks BBa_K2922034 (The Colicin-N operon under T7 promoter BBa_K525998 control), BBa_K2922035 (The Colicin-N operon under pBAD BBa_K206000 control), BBa_K2922036 (The Colicin-E1 operon under T7 promoter BBa_K525998 control) and BBa_K2922037 (The Colicin-E1 operon under pBAD BBa_K206000 control) were built and transformed into E.coli BL21 (DE3) successfully, which conformed by colony PCR and sequencing (Fig. 15).
Fig. 15. Colony PCR in 1% agarose gel. (A) The target bands of BBa_K2922037 (about 2400 bp), (B) The target bands of BBa_K2922035 (about 2100 bp), BBa_K2922036 (about 2300 bp) and BBa_K2922034 (about 2000 bp).
The E.coli BL21 (DE3) holding corresponding plasmids were cultured and expressed colicin kit through induction. The culture medium was collected as the sample for inhibition zone experiment, of which the protocol can be viewed in Notebook-Experiment-Inhibition zone. The virulence of Colicin-N and Colicin-E1 were verified by measuring the size of inhibition zone between the experimental and control group.
Fig. 16. The inhibition zone experiments through Oxford cup method. The results of experimental groups (induced) and the control groups (non-induced) were shown in the left and right in every petri dish respectively. (A) TN, namely the inhibition zone of strains expressing Colicin-N kit regulated by T7 promoter. (B) PN, namely the inhibition zone of strains expressing Colicin-N kit regulated by pBAD (arabinose promoter). (C) TE, namely the inhibition zone of strains expressing Colicin-E1 kit regulated by T7 promoter. (D) PE, namely the inhibition zone of strains expressing Colicin-E1 kit regulated by pBAD (arabinose promoter).
The secretion of Colicin-N and Colicin-E1 promoted by Nkil and Ekil
The E.coli BL21 (DE3) holding BioBricks BBa_K2922034 and BBa_K2922036 were cultured and expressed by adding corresponding inducer. The supernatant was obtained after centrifuging at 13000 rpm and used as the sample of inhibition zone experiment, of which the protocol can be viewed in Notebook-Experiment-Inhibition zone. The secretion of Colicin-N and Colicin-E1 was analyzed by measuring the size of the inhibition zone.
Finally, we found that the sizes of inhibition zone in the experimental group were larger than that in the control group (Fig. 17) and the data were recorded (Tab. S2).
Fig. 17. The inhibition zone experiments through Oxford cup method. The results of experimental groups (induced) and the control groups (non-induced) were shown in the left and right in every petri dish respectively. The supernatant was used as sample in each group, which obtained from corresponding culture medium. (A) TN sup, namely the inhibition zone of strains expressing Colicin-N kit regulated by T7 promoter. (B) TE sup, namely the inhibition zone of strains expressing Colicin-E1 kit regulated by T7 promoter.
Brief Summary of Aggressive
Globally, we can say that our cloning was successful although we met some troubles in growing the resulting transformed bacteria. We successfully demonstrate the virulence of Colicin-E1 and Colicin-N, and expressed proteins needed in our design verified by SDS-PAGE or spectrophotometer. In view of all of this, we finally completed the design and demonstrate its designed function primarily.
Reference
1. G. Xie, et al., Genome sequence of the cellulolytic gliding bacterium Cytophaga hutchinsonii. Appl Environ Microbiol 73, 3536-3546 (2007).
2. http://parts.igem.org/Part:BBa_K1610300
3. S. S. Lakhundi, Synthetic biology approach to cellulose degradation Doctoral Dissertation, The University of Edinburgh, UK, 2012.
4. K. O. Duedu, C. E. French, Characterization of a Cellulomonas fimi exoglucanase/xylanase-endoglucanase gene fusion which improves microbial degradation of cellulosic biomass. Enzyme Microb Technol 93-94, 113-121 (2016).
5. M. Crandall, et al., Temperature-Sensitive Mutants of Escherichia coli Affecting β-Galactoside Transport. J Bacteriol 105, 609-619 (1971).
Fig. S1. Gene circuit involved in protein expression and SDS-PAGE analysis of YebF-CenA by silver staining. (A) Gene circuit was constructed (BBa_K2922007) to express YebF-CenA. (B) Target bands of YebF-CenA (red arrow, 60 kDa). The character “R” stands for the flexible linker.
Fig. S2. Gene circuit involved in protein expression and SDS-PAGE analysis of YebF-Cex by Coomassie blue staining. (A) Gene circuit was constructed (BBa_K2922008) to express YebF-Cex. (B) Target bands of YebF-Cex (red arrow, 60 kDa). The character “R” stands for the flexible linker.
Fig. S3. Gene circuit involved in protein expression and SDS-PAGE analysis of CenA (114-kil-) by silver staining. (A) Gene circuit was constructed (BBa_K2922017) to express Kil and CenA. (B) Target bands of CenA (blue arrow, 47 kDa) in a brighter graph. (C) Target bands of CenA (red arrow, 47 kDa) in a darker graph.
Fig. S4. Gene circuit involved in protein expression and SDS-PAGE analysis of CenA (109-kil-) by silver staining. (A) Gene circuit was constructed (BBa_K2922021) to express Kil and CenA. (B) Target bands of CenA (red and blue arrows, 47 kDa).
Fig. S5. Gene circuit involved in protein expression and SDS-PAGE analysis of CenA (112-kil-) by silver staining. (A) Gene circuit was constructed (BBa_K2922016) to express Kil and CenA. (B) Target bands of CenA (red and blue arrows, 47 kDa).
Fig. S6. Gene circuit involved in protein expression and SDS-PAGE analysis of Cex (114-kil-) by silver staining. (A) Gene circuit was constructed (BBa_K2922020) to express Kil and Cex. (B) No target bands on the gel.
Fig. S7. Gene circuit involved in protein expression and SDS-PAGE analysis of Cex (109-kil-) by Coomassie blue staining. (A) Gene circuit was constructed (BBa_K2922018) to express Kil and Cex. (B) Target bands of Cex (red and blue arrows, 47 kDa).
Fig. S8. Gene circuit involved in protein expression and SDS-PAGE analysis of Cex (112-kil-) by Coomassie blue staining. (A) Gene circuit was constructed (BBa_K2922019) to express Kil and Cex. (B) Target bands of Cex (red and blue arrows, 47 kDa).
Fig. S9. Quantitative analysis for the enzymatic activity of YebF-Bgl1A and Bgl1A (109/112/114-kil-) was supported through HPLC. (A, C, E, G) The concentration of cellobiose and glucose of sup at different times, for different experimental and control groups respectively. (B, D, F, H) The concentration of cellobiose and glucose of broken sup at different times, for different experimental and control groups respectively.
Tab. S1. The calculation results of consuming and producing rate
Tab. S2. The results of the inhibition zone experiments
