Difference between revisions of "Part:BBa K3924045"

Latest revision as of 23:45, 21 October 2021

mcmK-mcmL polycistrons with Ptac lacO promoter

mcmK-mcmL polycistrons with Ptac lacO promoter

Sequence and Features

Profile

Name: mcmKL
Base Pairs: 2417bp
Origin: Escherichia coli, merge mcmK and mcmL
Properties: A polycistron can express both McmK and McmL. MchK and MchL can modify the precursor of MccH47.

Usage and Biology

The mcmK and mcmL are both from E.coli CA46. The mcmK encodes McmK, which can modify the precursor of the Microcin H47(MccH47) . The mcmL encodes McmL, which can modify the precursor of MccH47.^[1] The polycistron aim to make the engineering bacteria have the ability to modify precursor of MccH47.

Design and Construction

To construct the plasmid, we use endonuclease SpeI to cut RGP-mcmK at 2bp after mcmK open reading frame(ORF), then we used NEB Hifi DNA assembly to add RBS(ribosome binding site)-mcmL between mcmK and rrnB terminator.

Figure 1: The design of mcmKL

Functional Verification

We did western blotting(WB) to test whether the polycistron can express mcmL and mcmK successfully and we succeed.The time was the boiling sample time in WB.

Figure 2: The Western Blotting result

The whole story

Adherent-invasive Escherichia coli (AIEC) is one of the bacteria reported to be involved in IBD pathogenesis. As its name suggests, AIEC can first adhere to intestinal epithelial cells and microphages and then invade them. This would elicit inflammatory responses in intestine. We planned to introduce the mature microcin expression system of E.coli CA46 into E.coli Nissle 1917 to produce functional microcin, so that it can inhibit AIEC in the intestine of IBD patients and help restore their gut microflora composition^[2].
E.coli CA46, another E.coli strain capable of producing mature MccH47, has 9 genes in its MccH47 expression system: mchB, which encodes the precursor; mcmK, mcmL and mchD, which are necessary for the post-transcriptional modificaiton of the precursor; mchE and mchF, which transport MccH47 from the cytosol to the outside environment; mchC and mchX, whose functions are still unclear but can regulate the expression of MccH47; mchI, which encodes an inhibitory peptide^[3] that protects the producer itself. We found that although WT EcN already possesses homologs for all 9 genes mentioned above, these genes are divided into two cassettes in its genome, with mcmK and L located in a different cassette from the other 7 genes. This led us to speculate that they may be regulated separately, and that we may not need to introduce the entire set of 9 genes to achieve controllable production. Therefore, we designed two approaches. Approach I is to simplify the expression system and see if it can work with fewer genes. Approach II is to introduce all 9 genes at once on one plasmid. This latter approach has been proven practical by Jacob D. Palmer’s work in 2018, in which E. coli Nissle 1917 successfully obtained this system and synthesized mature MccH47.

Approach Ⅰ

We designed two simplified systems to test whether they can make E. coli Nissle 1917 produce MccH47.
Our first system contains only mchB and mchI, merged as a polycistron that has two ribosome binding sites and a single promoter. (Fig.1)The polycistron was assembled into an RGP plasmid, with Ptac-lacO as the promoter and rrnB as the terminator. For the convenience of further verifications, we made two versions of RGP-mchIB, with and without His tag.
Our second system contains mchB, I, K and L as two polycistrons, namely mchIB and mcmKL, each constructed in the same manner as in the first system.

Approach Ⅱ

Although this approach is to straightforwardly express all 9 genes on one plasmid, simply fusing these genes together will not work. Such a recombinant plasmid would be too big, and would also contain too many promoters, which can lay excessive burden on the chassis bacteria. So, based on the original arrangement of the 9 genes involved, we put them into four polycistrons: mcmK-mcmL, mchI-mchB-mchX, mchC-mchD, mchE-mchF. We had constructed them successfully.(Fig.3)For polycistrons containing the regulatory genes(mcmK-mcmL, mchC-mchD, mchE-mchF), we placed them under a constitutive expression promoter -- J23119. For mchI-mchB-mchX, we used Ptac-lacO to represent a regulatory promoter. Unfortunately, we failed to construct the final version plasmid, as the mcmKL failed to merge with other polycistrons.(Fig.4)(We added other three polycistrons at the basis of polycistron mchIBX. Therefore, we didn't identify whether the mchIBX existed in the huge plasmid via PCR.)

Figure 3: The identification of four polycistrons in DNA level

Figure 4: The identification of four polycistrons in DNA level

WB

We did western blotting(WB) to test whether the polycistron can express the proteins successfully. Fig.5 showed that polycistron mchIB had expressed MchI and MchB. However, maybe because both of the them are small peptides, the bands could not be separated successfully in the WB. As for mchIB-mcmKL, mcmK and mcmL succeed. However, mchI and mchB maybe fail for some reason.

Figure 5. The WB of polycistron mchIB and mchIB_mcmKL

Fig.6 showed that polycistron mchCD, mcmKL, and mchIBX had expressed the corresponding proteins successfully. As for mchEF, because it was too large, we had problem in introducing it into stbl3 and thus we coundn't conduct WB of mchEF.

Figure 6. The WB of polycistron mchIBX, mchCD and mcmLK

Co-culture assay

To demonstrate the competitive and antimicrobial ability of the microcin-producing strain, we co-cultured our engineered strain and a susceptible E.coli DH5α strain (imitating pathogen strain AIEC) that expresses EGFP, and then tested the green fluorescence with FACS to show whether co-culturing with our microcin-expressing strain had inhibited the growth of the susceptible strain. First, we examined the competitive ability of strains EcN-IB (EcN with MchIB expressed) and EcN-IB-His (EcN with MchIB added with a His-tag expressed) (Fig.7).

Figure 7.FACS result of co-culture experiment of EcN strains EcN-IB and EcN-IB-His

Result indicates that EcN expressing MchB and MchI has succesfully presented antimicrobial activity against DH5α, whether the protein contains a His-tag or not. If more repeated experiments support our results, we can draw the conclusion that mchB and mchI alone are sufficient for producing antimicrobial activity to a degree. Next, to investigate if an optimal dosage exists when we apply our microcin-producin strain, we test whether the antimicrobial activity depends on the initial ratio between our engineering strain and susceptible strain (Fig.8).

Figure 8.FACS result of co-culture experiment of EcN strains EcN-IB, EcN-IB-His and DH5α strain with different initial ratio. Red data represent to inhibition percentage of the corresponded strain, calculated by GFP-high cell ratio in treatment group devided by that in negative-control group.

As shown in Fig.9, no obivous dependency was observed. Maybe repeated tests are required for a more stable result with staticsically significance. Then, we considered if introducing mcmK and mcmL can enhance the antimicrobial activity of microcin (more precisely,the precursor of microcin MccH47) produced by mchB, for those two are crucial genes for the processing of MccH47. Same co-culture experiment has been accomplished to test the activity of the microcin produced by EcN strain EcN-IBKL (expressing MchB, MchI, McmK and McmL) and EcN-IBKL-His (with all exogenous protein added with His-tag) (Fig.9).

Figure 9: The FACS result of mchIBKL

Our FACS result indicates that EcN expressing MchI, MchB, McmK and McmL (as well as proteins with His-tag) has presented relatively stronger antimicrobial activity than EcN with only mchI and mchB expressed. This suggest that expressing mcmK and mcmL should be an effective way to enhance the antimicrobial activity. However, EcN-IBKL-His seems to have higher antimicrobial activity than EcN-IBKL, which is hard to explain because the His-tag should be a potential disruptor of protein activity. This suggests potential error in our experiments and repeated tests are required for a more stable and convincing result.
The result suggests that simply overexpressing microcin precursor in EcN can already make it capable of inhibiting DH5α to an extent, which means simplification of the expression system is indeed possible. Also, introducing extra modification factors of microcin precursor is an effective way of enhancing the performance of microcin.

Zone of inhibition test

Zone of inhibition test is usually a gold standard to test whether an agent exhibits antimicrobial activity and therefore a good choice for further validation of our microcin activity.

Figure 10. Protocol for zone of inhibition test. The red X and Y are conditions wait to be confirmed in the pre-test

A pre-test with kanamycin (Kan, 10 × working concentration) as antimicrobial substance was conducted to determine the optimal experimental condition (Fig.10), including the concentration of agar (X in Fig.10) and susceptible bacteria (Y in Fig.10). The result showed that agar concentration is the most crucial aspect to this assay whereas the cell concentration is less important. The optimal agar concentration should be 0.5%.(Fig.11)

Figure 11. Result of pre-test for zone of inhibition test. Agar concentration corresponds to X in Fig.10, and OD600 corresponds to Y in Fig.10.

Due to the time limit, zone of inhibition test for microcin hasn't been accomplished.

Future Plan

We will continue to construct the plasmid in Approach II and will test if it can endow EcN with antimicrobial activity. Zone of inhibition test will be conducted to examine the activity of microcin produced by each strain.
In addition, since we are currently using lacO as the sensing module for convenience, we will complete our design by replacing it with other sensing systems specific to IBD treatment, like sensors for Tetrathionate(ttr) or NO, both of which are indicators of inflammation.

Reference

[1] Vassiliadis G, Destoumieux-Garzón D, Lombard C, Rebuffat S, Peduzzi J. Isolation and characterization of two members of the siderophore-microcin family, microcins M and H47. Antimicrobial Agents and Chemotherapy. 2010 Jan;54(1):288-297.
[2] Lee, J. G., Han, D. S., Jo, S. V., Lee, A. R., Park, C. H., Eun, C. S., & Lee, Y. (2019). Characteristics and pathogenic role of adherent-invasive Escherichia coli in inflammatory bowel disease: Potential impact on clinical outcomes. PloS one, 14(4), e0216165.
[3] Palmer, J. D., Piattelli, E., McCormick, B. A., Silby, M. W., Brigham, C. J., & Bucci, V. (2018). Engineered Probiotic for the Inhibition of Salmonella via Tetrathionate-Induced Production of Microcin H47. ACS infectious diseases, 4(1), 39–45.