Team:XMU-China/Demonstrate
In order to characterize the mutual beneficial part of our design among bacterial communities, we plan to co-culture the engineering bacteria carrying plasmids containing cex and cenA genes with cellulose as the sole carbon source, and measure their growth statuss to evaluate whether our design works in order.
1. Co-culture experiment with cellulose filter paper
In the previous experiments, we measured the expression intensity of different promoters including J23109 (BBa_J23109), J23112 (BBa_J23112), J23114 (BBa_J23114) and PT7 (BBa_K525998), and measured their intracellular and extracellular activities. For this part, you can find more information at Experiment.
We finally decided to take J23109(BBa_J23109) as our promoter. Because it showed the best protein expression intensity in previous experiments. Finally, we transformed the plasmids pSB1C3 carrying J23109-kil-PT7-cenA-PT7-bgl1A (BBa_K2922021) and J23109-kil-PT7-cex-PT7-bgl1A (BBa_K2922022) into E.coli BL21 (DE3).
In addition to the two plasmids mentioned above, we also transformed another biobrick PT7-RBS (BBa_K525998) into E.coli BL21 (DE3) and we designed experiment based on these three strains. The experimental groups and the control group are listed below.
Tab. 1. The co-culture of the cocultivation growth curve.
The strains of each group are named as the codes of the genes carried. Click the gene for more details.
| Experimental group (cellulose filter paper) | Control group (glucose) | Blank group(cellulose filter paper) | |
|---|---|---|---|
| Codes in Lab | C1(cex+cenA) | G1(cex+cenA) | B(pure cellulose filter paper) |
| C2(cenA) | G2(cenA) | ||
| C3(cenx) | G3(cenx) | ||
| C4(PT7-RBS) | G4(PT7-RBS) |
Fig. 1. The culture result of the co-culture experiment with cellulose filter paper.
The protocol of the experiment can be viewed in the Experiment. The growth curve is shown in the figure below.
Fig. 2. The growth curves of the co-culture experiment with cellulose filter paper.
These results show that the results in the co-culture group (C1) are caused by the cooperation between different engineering bacteria, which is in line with our initial design.
However, cellulose filter paper will break into insoluble fine fibers both physically and chemically in the process of shaking culture, and it will precipitate to the bottom of 96-well plate when we use the enzyme scale instrument to measure the OD600 value. Due to that, it is difficult to collect accurate data, which directly results in a large systematic error.
And in this experiment, we surprisingly found that the E.coli carrying both cenA and cex can grow separately in the medium with cellulose filter paper as the sole carbon source, and the growth condition of group carrying cenA (C2) is better than that carrying cex (C3), even better than the co-culture group (C1).
2. Co-culture experiment with microcrystalline cellulose (MCC)
After that, in order to determine whether the growth of the E.coli in the C1 group is the contribution of two bacteria or a single one, we transformed the plasmid carrying J23109-kil-PT7-cex-PT7-bgl1A-PT7-mRFP (BBa_K2922023) into E.coli BL21 (DE3). This time, we also added a group of E.coli BL21 (DE3) that carrying J23109-kil-PT7-cenA (BBa_K2922015) (no bgl1A in this circuit) to control variables. Moreover, considering the crushing of cellulose filter paper, we replaced cellulose filter paper with microcrystalline cellulose (MCC) with higher dispersibility. The experimental groups and the control group are listed below.
Tab. 2. The co-culture of the cocultivation growth curve.
The strains of each group are named as the codes of the genes carried. Click the gene for more details.
| Experimental group (MCC) | Control group (glucose) | Blank group(MCC) | Blank group(glucose) | |
|---|---|---|---|---|
| Codes in Lab | C1(cex-bgl1A-mRFP+cenA-bgl1A) | G1(cex-bgl1A-mRFP+cenA-bgl1A) | B1(pure MCC) | B2(pure glucose) |
| C2(cenA-bgl1A) | G2(cenA-bgl1A) | |||
| C3(cex-bgl1A) | G3(cex-bgl1A) | |||
| C4(cenA) | G4(cenA) | |||
| C5(cex-bgl1A-mRFP) | G5(cex-bgl1A-mRFP) | |||
| C6(109-kil) | G6(109-kil) |
The protocol of this experiment is the same as the previous one, which could be viewed in Experiment.
After continuous measurement for 50 hours, the growth curves and fluorescence intensity curves are shown in the figure below (Fig. 4-5).
Fig. 3. The culture result of the co-culture experiment with MCC.
Fig. 4. The growth curves of the co-culture experiment with MCC.
Fig. 5. The fluorescence intensity curves of the co-culture experiment with MCC.
These results show that bacteria grow in all our groups. According to the prescription of our M9 culture medium, these bacteria are our engineering bacteria. The results of co-culture group (C1) shows that the engineering bacteria can grow in co-culture system, but it is not strong enough to prove that co-culture has obvious advantages compared with culture separately.
Because microcrystalline cellulose (MCC) can not be dissolved in water, the culture system is still a suspension, just like before. This also brings great error to our result. This sort of error is caused by the non-repeatability of sampling. Although we try our best to suspend the culture medium evenly before each sampling, and also use the same sampling method, the differences between the samples are still very significant. Another important reason is the sedimentation of MCC particles. This brings unpredictable systematic error to the measurement of OD600 value.
3. Spread plate experiment with serial dilution
Because the direct characterization by measuring OD600 and fluorescence intensity has failed to prove our design, and there are some phenomena that we didn't expect or even can't explain in the experiment process, such as the growth condition of the group carrying cenA (C2) in cellulose medium was better than that in co-culture system (C1), and the blank group (B1) stimulated higher fluorescence intensity than all experimental groups (C1-C6) at 586 nm, etc.
We took some medium (C1,C2,C5) for serial dilution and then we get some single colony by spread plate method. The results are shown in the figure below.
Fig. 6. The results of spread plate after culture.
Unfortunately, we did not observe any colony turning red, even after a long period of culture.
4. Plasmid sequencing
Finally, we sequenced the plasmid used to construct J23109-kil-PT7-cex-PT7-bgl1A-PT7-mRFP (BBa_K2922023). Interestingly, the results showed that a 188 bp fragment in the coding sequence of one cex sample was missing. We highly speculated that the deletion occurred in the molecular cloning process. At the same time, another sample carrying cex was found to have an extra base pair in its coding sequence, which results in a frameshift mutation. We think these are possible explanations for why our results are different from our expectation. At the same time, we suspect that there are still some unknown factors in our experiment, which together lead to the failure of our characterization.
Fig. 7. The missing fragment in the coding sequence of cex sample.
Fig. 8. The extra base pair in the coding sequence of cex sample.
Before we could investigate how the “aggressive” part work among bacteria groups, we need to examine the reliability of our two inducible systems: the PT7-RBS promoter(BBa_K525998) and the arabinose promoter (pBAD) (BBa_K206000). Inhibition zone test was carried out to verify the virulence of Colicin-E1 and Colicin-N and inducibility of PT7-RBS and pBAD promoters. The protocol can be viewed in Notebook-Experiments- Inhibition zone, and the data are listed below (Fig. 9).
Fig. 9. Results of inhibition zone test for strains carrying. (A) BBa_K2922034, (B) BBa_K2922035, (C) BBa_K2922036 and (D) BBa_K2922037. Area marked by a tick is experimental group that has induced by lactose or arabinose and area marked by a cross is control group.
These results not only shown the virulence of Colicin-E1 and Colicin-N but also verified the inducibility of T7 or pBAD promoter, although the inhibition zone exist in control group indicate that these promoters may leaky.
Biobricks BBa_K2922038 (TND, namely the Colicin-N operon under T7 promoter control with a gfasPurple chromoprotein reporter) and BBa_K2922039 (PEF, namely the Colicin-E1 operon under pBAD control with an amajLime chromoprotein reporter) are designed to investigate how the aggressive part work among bacteria groups. We transformed plasmids pSB1C3 carrying these two biobricks and other two biobricks BBa_K1033914 and BBa_K1033917 into E.coli BL21 (DE3). Meanwhile, a series of growth curve experiments were designed (Tab. 3).
Tab. 3. The co-culture of the cocultivation growth curve. The strains of A and B are named as the codes of the biobricks carried. “+” means “induced” and “-“ means “non-induced”.
| Group | StrainA | StrainB | |
|---|---|---|---|
| Codes in Lab | Ⅰ | BBa_K1033914 | none |
| ⅠI | BBa_K1033917 | none | |
| ⅠII | +TND | none | |
| ⅠV | +PEF | none | |
| V | +TND | BBa_K1033914 | |
| VI | -TND | BBa_K1033914 | |
| VII | +PEF | BBa_K1033917 | |
| VIII | -PEF | BBa_K1033917 | |
| IX | +TND | +PEF | |
| X | -TND | +PEF | |
| XI | -TND | -PEF | |
| XII | +TND | -PEF |
The protocol can be viewed in Notebook-Experiments-Growth Curve, and the data are listed below (Fig. 10-11).
Fig. 10. The growth curves in OD value. (A) The curves about group Ⅴ (B) The curves about group Ⅵ (C) The curves about group Ⅶ (D) The curves about group Ⅷ.
Fig. 11. The OD600 Value curves. (A) The curves about group Ⅴ (B) The curves about group Ⅴ and Ⅵ.
These results indicate that interference from E.coli cell density to measured values of OD577 and OD458 is beyond our expectation. Therefore, before removing the interference, there are some problems in using chromoproteins to specifically record the growth curve of a strain in co-culture system. But the cell density in group Ⅴ (induced group) was significantly lower than group Ⅵ control group (non-induced group), which means the Colicin-N kit was active successfully by IPTG induced and indicator’s growth was inhibited. We need further processing of data to analyze the growth status. The results of the analyzation are listed below (Fig. 12-13) and the calculations are attached to the appendix.
Fig. 12. The relative OD Value growth curves. (A) The curves about group Ⅴ and Ⅵ. (B) The curves about group Ⅶ and Ⅷ.
Fig. 13. The relative OD Value growth curves of TND and PEF. (A) The curves of group Ⅸ. (B) The curves of group Ⅹ. (C) The curves of group ⅩⅠ. (D) The curves of group ⅩⅡ.
As the figures shows, the effect of Colicin-N kit is significant, which means E.coli Bl21 (DE3) carrying biobrick BBa_K2922038 (TND) possesses the ability to kill other bacteria in co-culture system and plays as an “Aggressive” part. However, the virulence of Colicin-E1 kit was not presented by the E.coli Bl21 (DE3) carrying biobrick BBa_K2922039 (PEF), according the figure above.