iGEM 2022 — Team gems-taiwan

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{% block title %}Notebook{% endblock %}
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# **Notebook**

* [**Notebook**](#dates)
  [**Protocol**](#protocol)

## Lab Notebook

### Here’s an overview on the accomplishments and challenges we had faced during our experiments.

| Dates | Results | Description & Notes | Figures |
| --- | --- | --- | --- |
| 7/7 | • Lysogeny broth (LB) made   • Agar Plates made | • Preparations for future experiments | N/A |
| 7/12 | • Concentration test | • Test to confirm whether old constructs can be used | N/A |
| 7/15 | • Transformed DH5a with PSB1C3 containing plasmid | • Diluted pSB1C3-pSTE12-18, pSB1C3-RFP, and pSB1C3-CmR according to the concentration tested on 7/12 for 1ng/ul transformation [1-1]   • Transformed and plate with DH5𝛼 (for plating we grow bacteria overnight 12~16 hrs) [1-2~1-5] | [7/15](https://static.igem.wiki/teams/4137/wiki/notebook/notes/7-15.png) |
| 7/16 | • Colonies grown in 7/15 were moved into LB | • SB1C3-CmR didn’t grow   • Selected colonies for the other two and grew them in liquid LB (30℃) | [7/16](https://static.igem.wiki/teams/4137/wiki/notebook/notes/7-16.png) |
| 7/17 | • Verified that pSB1C3 was contained | • Did digestion tests with different restriction sites to verify and extracted pSB1C3 (used RFP construct) [2]   • Extracted backbone (pSB1C3) and measured its concentration [1] | [7/17](https://static.igem.wiki/teams/4137/wiki/notebook/notes/7-17.png) |
| 7/29 | • First cloning cycle | • Resuspended ordered constructs (ccdA, ccdB, mleR-first half, chitinase-first half)   • Digested backbone on E-S restriction sites and ligated with each of our constructs individually   • Transformed and plate the ligated products into DH5𝛼, wait for colonies to grow overnight [1~4] | [7/29](https://static.igem.wiki/teams/4137/wiki/notebook/notes/7-29.png) |
| 8/3 | • Verification (first cloning cycle failed) | • Did colony PCR with primers   • All the bands are way too largely | [8/3](https://static.igem.wiki/teams/4137/wiki/notebook/notes/8-3.png) |
| 8/6 | • Second try for cloning cycle   • Redo cloning cycles for ccdA and ccdB | Improvements and discussion made   • 1ul Digestion Enzyme   • Verification of backbone and RFP band   • Reminder: heat shock before transformation   • Digested vector (pSB1C3-RFP) and extracted backbone   • Re-attempt cloning cycle (same steps as 7/29)   • Refilled LB | N/A |
| 8/7 | • Verified pSB1C3+chitinase & pSB1C3+mleR (failed)   • Redo cloning cycle for all constructs (third re-try) | • Colony PCR results didn’t look good [1]  • Re-attempt cloning cycle   • Digested and extracted vector and fragments [2]   • Overnight ligation | [8/7](https://static.igem.wiki/teams/4137/wiki/notebook/notes/8-7.png) |
| 8/8 | • Grow DH5𝛼 with our ligated products | • Transformed and plated the four ligated constructs with DH5𝛼 (thanks for Bryan’s assistance) | [8/8](https://static.igem.wiki/teams/4137/wiki/notebook/notes/8-8.jpg) |
| 8/12 | • Verifying for ligated construct (failed) | \*50x TAE buffer was accidently added   • decided to redo Colony PCR the next day (lost a few samples while loading) | N/A |
| 8/13 | • Chitinase-1st half success | • Redo Colony PCR   • One chitinase colony had the correct band   • Unfortunately other constructs where wrong | [8/13](https://static.igem.wiki/teams/4137/wiki/notebook/notes/8-13.png) |
| 8/14 | • Success colony grown in LB | • Moved successfully ligated chitinase colony into LB   • Grew 4 tubes of chitinase 1st half and one tube of vector with DH5𝛼 | N/A |
| 8/15 | • Vector and Chitinase first-half extracted | • Miniprep pSB1C3-RFP & pSB1C3-Chitinase first-half (colonies 1~4) | [8/15](https://static.igem.wiki/teams/4137/wiki/notebook/notes/8-15.png) |
| 8/18 | • Vector digested | • Prepare backbone for future cloning cycle [2]   • Gel was cutted and preserved in -20℃ fridge   • concentration was tested [1] | [8/18](https://static.igem.wiki/teams/4137/wiki/notebook/notes/8-18.png) |
| 8/19 | synthesize designed chitinase full construct | 3rd round cloning cycle   • Chitinase 2nd-half resuspension and digestion   • Gel electrophoresis & extraction   • Ligate Chitinase 2nd-half with pSB1c3-Chitinase 1st-half   • Transform and plate chitinase-full construct | N/A |
| 8/20 | Verify chitinase full construct (failed) | Colony PCR, bands didn’t match | [8/20](https://static.igem.wiki/teams/4137/wiki/notebook/notes/8-20.png) |
| 8/21 | • Verify chitinase full construct (succeed) • 3 plates of DH5𝛼, each contains one construct: pSB1C3-ccdA, pSB1C3-ccdB, pSB1C3-mleR-1st half | • Tried Colony PCR with the same plate again, but chose 1x more colonies this time to increase chance of obtaining our construct [1]   4th round colony cycle:   • Redo cloning cycle for the other constructs: psb1c3-ccdA, psb1c3-ccdB, psb1c3-mleR-1st half   • Digest fragments and vectors [2]-> concentration test   TE buffer misused for vector   • Did second vector digestion [3]-> gel extraction   ligate backbone with fragments   transform and plate with DH5𝛼 | [8/21](https://static.igem.wiki/teams/4137/wiki/notebook/notes/8-21.png) |
| 8/26 | • Same as 8/21 | • colony PCR-> gel electrophoresis [1]   \* Retransformation and plate previous ligation products but with recovering [2] | [8/26](https://static.igem.wiki/teams/4137/wiki/notebook/notes/8-26.png) |
| 8/27 | • Cloning Cycle (Success)   • Grew bacterias that contain our constructs in LB | • Colony PCR— ALL BANDS CORRECT  • Grow correct colonies into LB | [8/27](https://static.igem.wiki/teams/4137/wiki/notebook/notes/8-27.png) |
| 8/28 | • Constructs finally collected:   • pSB1C3-ccdA   • pSB1C3-ccdB   • pSB1C3-mleR-1st half | • Miniprep and concentration test | N/A |
| 8/29 | • Cloning cycle (success) for mleR-2nd half+pSB1C3-mleR 1st half (got the full mleR construct | 5th round Cloning cycle (for mleR full construct)   • Vector and fragment digestion [1]   • Run gel-electrophoresis and extracted backbone   • Leaved ligation overnight   For psB1C3-ccdA-His/ccdB-Myc -tag addition   • Q5 mutagenesis (PCR)   • Run gel for verification [2]   • KLD reaction-> transformed and plated with DH5𝛼 | [8/29](https://static.igem.wiki/teams/4137/wiki/notebook/notes/8-29.png) |
| 8/30 | • DH5𝛼 grown with (supposedly) mleR-full construct   • Starting for chitinase expression   • Grow colonies that contain tag-added constructs | For mleR-full construct:   • transform and plate yesterday’s ligation product   For Chitinase protein production:   • transformed and plated chitinase-full plasmid into BL21 | N/A |
| 9/1 | • Verification on the mleR full construct (success)   • Verification on whether if chitinase is successfully transformed (failed)   • Verification on tag-added constructs (success) | For mleR full construct   • Colony PCR bacteria grown yesterday [1]   • run gel electrophoresis [2]   • Grow colony that has been successfully transformed into LB   For Chitinase protein production: • Colony PCR   For psB1C3-ccdA-His/ccdB-Myc -tag addition • Colony PCR   Gel electrophoresis and grow correctly transformed colonies into LB | [9/1](https://static.igem.wiki/teams/4137/wiki/notebook/notes/9-1.png) |
| 9/2 | • Successfully collected mleR-full construct   • Verification on whether if chitinase is successfully transformed (success) | For mleR full construct   • Miniprep   For chitinase protein production:   • Retry Colony PCR with same plate   • Grow successfully transformed colony into LB | N/A |
| 9/3 | • Expressed and collected chitinase (waiting for verification) | \*Did concentration test for yesterday’s mleR miniprep   For chitinase protein production:   • Move small amount of colonies into starter culture   • OD measurement (check colony concentration if reached log phase)-> protein expression and extraction/collection   • Start Chitinase protein production   • 19:20 begin induction, OD 0.354 25C, 250rpm => sample collection (0h, 1h, 2h, 3h)   • 20:20 begin induction 2, OD 0.348 => sample collection (0h, 1h, 2h, 3h) | [9/4](https://static.igem.wiki/teams/4137/wiki/notebook/screen-shot-2022-10-14-at-02-22-10.png) |
| 9/4 | • Grow bacteria containing CcdB and mleR (individually) | For protein expression CcdB & mleR (two are separated)   • Transform and plate CcdB & mleR (BL21) [2] | [9/4](https://static.igem.wiki/teams/4137/wiki/notebook/notes/9-4.jpg) |
| 9/5 | • Collected tag added constructs (success) | For psB1C3-ccdA-His/ccdB-Myc -tag addition   • Miniprep and test concentration with spectrophotometer | [9/5](https://static.igem.wiki/teams/4137/wiki/notebook/notes/9-5.png) |
| 9/7 | • CcdA-6xHis E-S digestion quantification   • Got bacteria transformed with pSB1C3-mleR-ccdA-His | For psB1C3-ccdA-His/ccdB-Myc -tag construct building Cloning Cycle (6th)   • Digested pSB1C3-mleR-full construct   gel electrophoresis-> extraction-> Miniprep-> concentration test • Ligate mleR-full with CcdA-6xHis   Transform and plate | N/A |
| 9/8 | • Transformed separated and combined constructs   • Started starter culture-> ready for protein expression | For ccdA, ccdB, mleR and other combined constructs   • Cloning cycle & CPCR & plating: CcdA-mleR, Ccda-6xHis, CcdB-Myc tag   • Starter LB mleR, CcdB BL21 | N/A |

[Lab Results Pictures](https://static.igem.wiki/teams/4137/wiki/coculture-10-2-10-4-10-6-10-7/lab-photos.pdf)

## Protocol

To view the specific procedures for our experiments, please download the file attached below.

[Lab Protocol](https://static.igem.wiki/teams/4137/wiki/lab-protocol.pdf)

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# **Engineering**

* [Summary](#summary)
  [Cloning](#Cloning)
  [Colony PCR](#colonypcr)
  [Troubleshooting](#troubleshooting1)
  [Re-attempt](#reattempt1)
  [Re-attempt Colony PCR](#reattemptcolonypcr)[Protein Expression/Thermolysis](#thermolysis1)
  [Thermolysis](#thermolysis2)
  [Troubleshooting](#troubleshooting2)
  [Re-attempt](#reattempt2)
  [SDS-PAGE](#sdspage)

## Summary

We were able to successfully assemble all of our planned constructs with the exception of pSB1C3::CcdA::CcdB by analyzing the mode of failure and redesigning our cloning protocol to resolve identified issues.

For expression verification of our proteins, we created a simple and novel thermolysis protein extraction protocol as a means of overcoming equipment limitations, then successfully demonstrated the viability of this protocol. We resolved clumping issues during sample preparation and used SDS-PAGE to demonstrate that samples treated using this protocol produce interpretable and viable results.

For a comprehensive experimental overview of our project, please visit the  [Experiments page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D)..

## Cloning

Plasmid inserts were acquired by ordering the synthesis of our construct designs via IDT gblocks synthesis.

Vectors were prepared by digesting pSB1C3::BBa\_J04450 then performing gel extraction on the 2k bp band. Inserts were digested and then DNA-extracted without running gel electrophoresis, as the sequence fidelity of synthesized gene fragments is high. Quantification was performed on the extracted DNA, and calculations were made to approximate the 3:1 insert-to-vector ratio. The ligation product was transformed into DH5α; after heat shock and six minutes of incubation on ice, the mixture was then plated on LB plate supplemented with chloramphenicol for selection.

Cloning procedures for the chi18h8 and mleR construct were broken up into two stages due to errors during gblocks synthesis. The process is explained in the figure below.

![...](https://static.igem.wiki/teams/4137/wiki/diagram1.png)

![...](https://static.igem.wiki/teams/4137/wiki/diagram1b.png)

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**Figure 1a.**  Two-step cloning process for pSB1C3::Chi18h8.
**Figure 1b.**  Two-step cloning process for pSB1c3::mleR

Apart from excess colony number on some plates, plating results appeared visually normal for the most part during cloning for pSB1C3::chi18h8(1/2), pSB1C3::CcdA, pSB1C3::CcdB, and pSB1C3::mleR(1/2).

![...](https://static.igem.wiki/teams/4137/wiki/fig11.png)

![...](https://static.igem.wiki/teams/4137/wiki/fig22.png)

![...](https://static.igem.wiki/teams/4137/wiki/fig33.png)

![...](https://static.igem.wiki/teams/4137/wiki/fig44.png)

![...](https://static.igem.wiki/teams/4137/wiki/fig55.png)

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**Figure 2-6.**  Plates for pSB1C3::CcdA, pSB1C3::CcdB, pSB1C3::mleR(1/2), pSB1C3::chi18h8(1/2), and pSB1C3::chi18h8.

### Colony PCR

Colony PCR was performed using backbone-specific primers; they attach to the biobrick prefix and suffix sites to flank inserts. The results of PCR should thus be DNA bands with the same length as the insert.

Ten colonies were chosen over multiple colony PCR tests, and pSB1C3::BBa\_J04450 is used as the negative control. Unfortunately, the vast majority returned as negative. Multiple double PCR bands were observed, and there was a consistent and anomalous 1.5k bp band visible on many PCR results. Four of the chi18h18(1/2) (labeled “chi”) colonies displayed the correct ~800 bp band corresponding to chi18h8(1/2); the rest were entirely incorrect. We initially hypothesized this result to be caused by antibiotic degradation decreasing the effectiveness of the selection plate.

![](https://static.igem.wiki/teams/4137/wiki/fig7.png)

**Figure 7.**  Colony PCR results for pSB1C3::CcdA, pSB1C3::CcdB, pSB1C3::mleR(1/2), and pSB1C3::chi18h8(1/2)

Additional cloning cycles were performed to create pSB1C3::chi18h8  [(the two step cloning process illustrated in Figure 1a.)](#fig1), pSB1C3::CcdA, pSB1C3::CcdB, and pSB1C3::mleR(1/2), this time with freshly cast LB agar plates. Plating results for the chi18h8 yielded numerous colonies, but poor colony PCR positive rate - 1 out of 11 displayed the 1.8 kb insert band corresponding to the correct and complete chi18h8 construct.

The singular verified DH5α pSB1C3::Chi18h8 colony was grown in liquid culture; miniprep was then performed to extract pSB1C3::Chi18h8.

![](https://static.igem.wiki/teams/4137/wiki/fig8.png)

**Figure 8.**  Colony PCR results for pSB1C3::Chi18h8.

Results for the other three constructs were exceptionally poor; only two colonies grew on the pSB1C3::CcdA plate and one for pSB1C3::CcdB and pSB1C3::mleR. All PCR bands were identical to the negative control (BBa\_J04450).

![](https://static.igem.wiki/teams/4137/wiki/fig9.png)

**Figure 9.**  Colony PCR results for pSB1C3::CcdA, pSB1C3::CcdB, and pSB1C3::mleR(1/2).

### Troubleshooting

Cloning cycles for pSB1C3::CcdA, pSB1C3::CcdB, and pSB1C3::mleR(1/2) failed. The erratic quantity of bacterial growth was inconsistent with digestion or ligation failure. Analysis of the failure mode demonstrated that failed bands appear to be of erratic, inexplicable sizes (1.5 kb, < 1 kb) that did not correspond to negative control pSB1C3(2k bp) or BBa\_J04450(1k bp) bands.

We thus hypothesized the underlying issue to be poor transformation efficiency resulting in low numbers of successfully transformed bacteria. To resolve this, we chose to include a recovery step by incubating the transformation mixture in LB to increase the number of viable bacteria.

### Re-attempt

Cloning cycles for pSB1C3::CcdA, pSB1C3::CcdB, and pSB1C3::mleR were redone with the additional step of recovery. The colony quantity on the plates appears visually normal.

![...](https://static.igem.wiki/teams/4137/wiki/fig100.png)

![...](https://static.igem.wiki/teams/4137/wiki/fig200.png)

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**Figure 10-12.**  Plating results for pSB1C3::CcdA, pSB1C3::CcdB, and pSB1C3::mleR(1/2)

### Re-attempt Colony PCR

Five colonies were chosen from each plate for colony PCR verification; pSB1C3::BBa\_J04450 was again used as the negative control. No dual bands were observed, and the success rate was significantly higher. Four of the pSB1C3::CcdA bands matched with the ~300 bp insert size, at least 3 of the pSB1C3::CcdB bands matched with the ~400 bp insert size, and all of the pSB1C3::mleR(1/2) bands matched with the ~600 bp insert size.

![...](https://static.igem.wiki/teams/4137/wiki/fig133.png)

![...](https://static.igem.wiki/teams/4137/wiki/fig144.png)

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**Figure 13-14.**  Colony PCR results for pSB1C3::CcdA, pSB1C3::CcdB, and pSB1C3::mleR(1/2).

The massive improvement in success rate after choosing to perform recovery demonstrates the effectiveness of the method and the validity of our hypothesis.

All verified construct colonies were then grown in liquid culture for plasmid amplification; miniprep was then performed to extract the constructs.

The redesigned protocol was applied to successfully assemble the following constructs: pSB1C3::CcdA, pSB1C3::CcdB, pSB1C3::mleR, pSB1C3::Chi18h8, pSB1C3::CcdA::mleR. These constructs were then transformed into BL21(DE3) for expression.

## Protein Expression/Thermolysis

Chi18h8 expression was performed in 50 ml of expression medium with BL21(DE3) E. coli. During literature research, it seemed apparent that expression of the protein required very particular protocols; it was thus decided that we’d follow Berini et al.’s protocol at all points if possible. Chi18h8 expression was performed at two temperatures, 37 oC and 30 oC; the former was used as a reference for standard in vitro settings, and the latter was used to simulate soil temperature. 5 ml of expression medium was used for cell harvest at time intervals of 2h and 4h. For CcdB and mleR, cell harvest was performed at time intervals of 0h, 0.5h, 1h, 2h, 3h, and 4h. For CcdA, cell harvest was performed at 3h. Expression protocol mostly followed Berini et al.’s methods.

### Thermolysis

However, we hit a roadblock at protein extraction due to a lack of equipment for performing sonication and chemical lysis. To resolve this, we formulated a thermolysis method based on tips from Lab 106 personnel and theory. Phosphate Buffered Saline (PBS) and 4X Sample buffer were added to pelletized cells in a volume ratio of 3:1, the total fluid volume was initially adjusted to 100 ul, and the cells were resuspended. This process was performed on CcdB and mleR samples.

It was noticed immediately that for larger pellets, the resuspended mixture became highly viscous, and clumping was observed in some cases. After heating the mixture at 95oC for 10 minutes, centrifuging at 4oC, 16000g, the supernatant was extracted and transferred to a new eppendorf tube; in multiple cases, a clump was left over. This also had the effect of significantly impacting the supernatant yield - only a few bubbles could be extracted from the thermolysed cell mixture.

![](https://static.igem.wiki/teams/4137/wiki/fig155.png)

**Figure 15.**  Clumping observed in resuspended cell pellets during thermolysis protocol.

### Troubleshooting

After consulting our instructor on this odd occurrence, she suggested it could be due to high concentrations of genomic DNA resulting in clumping. To resolve this issue, we decided moving forward to use double the total volume, i.e. 200 ul, to resuspend pellets. We hypothesized that this should dilute the genomic DNA and reduce clumping.

### Re-attempt

Protein samples that underwent clumping were salvaged with 100 ul extra PBS, then reheated to 95 oC for 10 minutes to break up the clump.

The heated eppendorf tubes were inspected mid-heating, and the clumps were observed to have somewhat dissolved. After the full 10 minutes, the clumps mostly disappeared. The mixture was re-centrifuged, and the supernatant was again transferred to a new eppendorf tube.

### SDS-PAGE

20 ul of the extracted proteins were taken and analyzed via SDS-PAGE.

![...](https://static.igem.wiki/teams/4137/wiki/fig166.png)

![...](https://static.igem.wiki/teams/4137/wiki/fig177.png)

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**Figure 16-17.**  Colony PCR results for pSB1C3::CcdA, pSB1C3::CcdB, and pSB1C3::mleR(1/2).

For both mleR and CcdB, correct protein bands are visible to a reasonable degree.

![...](https://static.igem.wiki/teams/4137/wiki/home-page-diagrams/18.png)

![...](https://static.igem.wiki/teams/4137/wiki/home-page-diagrams/19.png)

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**Figure 18-19.** SDS-PAGE of Chi18h8 and CcdA with mleR.

Chi18h8 and CcdA-mleR samples were thermolysed with the new 200 ul resuspension volume protocol, and essentially no clumping occurred.

For Chi18h8, protein bands were obscured by excessive protein quantity and excessive dyeing, but they are nevertheless distinguishable. For CcdA-mleR, protein bands were no longer distinguishable below 17 kDa; however, we believe this to be caused by the buffer system we were using. Because Tris-Glycine is poor at resolving protein bands smaller than 15 kDa, it is natural that CcdA(9.5kDa) would be hard to distinguish. Apart from that, correct, distinct mleR bands at ~35kDa were visible throughout all trial groups.

The extracted proteins were used for expression verification only due to time constraints. Since SDS-PAGE analysis demonstrates that proteins of interest were successfully extracted via thermolysis, we are able to conclude that our protocol for thermolysis is viable as a method of protein extraction for expression verification purposes.

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# **Experiments**

* [Experimental Overview](#Experimental-Overview)
* [Construct Designs & Cloning](#Construct-Designs-&-Cloning)
  + [Chitinase Secretion Construct](#Chitinase-secretion-construct)
  + [CcdA-CcdB Malate-induced Toxin-antitoxin Kill Switch Construct(s)](#ccdaccdb)
  + [Cloning Plans](#Cloning-Plans)
* [Protein Expression](#Protein-Expression-Purification-and-Functional-Assays)
  + [Expression of ccdA-ccdB Antitoxin System](#expressionccdaccdb)
  + [Chitinase Production](#Chitinase-Production)
* [Functional Assays](#functionalassays)
  + [Chitinase Secretion](#chitinasesecretion)
  + [Co-culture/Chitinase Activity Assay](#Co-culture/Chitinase-Activity-Assay)
  + [CcdA Induction Expression](#CcdA-Induction-Expression)
* [References](#references)

## Experimental Overview

To give our engineered bacteria antifungal activity against FOC TR4, we devised a chitinase secretion system, and designed a Chi18h8 secretion construct for said system. In order to ensure adequate biocontainment for our engineered bacteria, we designed a Toxin-Antitoxin kill switch system and three constructs to achieve that function - a CcdA-producing construct(BBa\_K4137012), CcdB-producing construct(BBa\_K4137013), and a mleR-producing construct(BBa\_K4137014). These constructs act as modular parts that can be assembled in various combinations to determine systemic behaviors.

The chitinase secretion system uses an unspecific effector that has the potential activity against non-FOC fungus, so the kill switch system will help us rein in the unintended environmental consequences of our low effector specificity. For both systems, we’ve performed multiple cloning cycles to construct our desired plasmids, verified protein expression in BL21 E. coli, and analyzed the results via SDS-PAGE.

For further investigations on whether our designed constructs perform their expected function, we plan to conduct several assays. For confirmation on the functionality of chitinase and extracellular secretion, we ran chitinase secretion and yeast co-culture assays. For identification of the correct co-production of both the toxin and antitoxin in bacteria, we’ll perform CcdA/CcdB expression assay. Induction expression will also be carried to confirm that the transcriptional activator pmleS (BBa\_K4137005) operates correctly in conjunction with mleR to induce production of CcdA. To relate the amount of malate with bacterial survival and growth and test the effectiveness of our kill switch, we’ll perform the drop assay to see bacteria survival outcomes in various environmental malate concentrations.

## Construct Designs & Cloning

### Chitinase secretion construct

The specific chitinase we selected is Chi18h8 – through literature review, many authors have concluded it as having potential applications in antifungal applications for crop disease biocontrol. Most crucially, Chi18h8 was shown to have antagonistic activity against Fusarium oxysporum in Hjort et al.’s analysis.

![Figure 1.](https://static.igem.wiki/teams/4137/wiki/chitinase.png)

**Figure 1.** BBa\_K4137009: Chitinase Secretion construct

For our design, we used a T7 promoter and T7 terminator to ensure high transcription levels of our gene of interest by E. coli. Along with the strong promoter and terminator, we included a strong ribosome binding site (RBS) to further optimize the constitutive protein production rate.

Since our goal is to have an extracellular secretion of Chi18h8 using the Sec secretion system, we fused Chi18h8 with a yebF secretion tag, which facilitates the movement of chitinase through the periplasm and into the extracellular medium. The YebF tag is a N-terminal peptide tag that is used to permit extracellular secretion of a protein of interest through protein fusion; the tag is cleaved off when the protein is secreted into the extracellular medium. We’re testing this in E. coli as a proof of concept for chitinase secretion before moving on to handling B. subtilis. We’ve also inserted a flexible glycine-serine linker (GS) between the two genes to stabilize our fusion protein.

To verify the successful production and secretion of the fusion protein of Chi18h8 and yebF tag, we inserted a 6xHis tag at the end of our construct design to help us isolate the protein in downstream protein purification assays.

Purification of the protein will allow us to experiment with various concentrations to determine what amount of chitinase is required to achieve sufficient inhibition of FOC; we can then use this information downstream to tweak the construct designs to optimize FOC inhibition.

### CcdA-CcdB malate-induced toxin-antitoxin kill switch construct(s)

The aim of the kill switch system is to constrain the activity of our engineered probiotic B. subtilis to the permissive environment that is the banana root rhizosphere so that it wouldn’t spread and cause unpredictable harm; the rhizosphere supplies sufficient malate to the bacteria through root exudates.

The kill-switch is based on the toxin-antitoxin pair CcdB/CcdA (BBa\_K4137000, BBa\_K4137001). As a DNA gyrase inhibitor, CcdB targets and traps DNA gyrase(GyrA), causing cell death; this toxicity is sequestered by antitoxin CcdA, which blocks the activity of CcdB by forming a CcdA-CcdB complex, preventing CcdB from binding into GyrA.

In order to control the survival of our bacteria and ensure that it would stay only in areas where malate is present in sufficient quantity, we used the mleR transcriptional activator and its respective regulon to control the expression of CcdA. The mleR regulon requires mleR to achieve maximal expression; without malate, mleR is not activated, and thus transcription via the mleR regulon is not induced, causing low CcdA production. If our bacteria moves beyond the banana root rhizosphere, the subsequent deficiency in CcdA will eventually lead to cell death due to the constitutive production of CcdB.

a. mleR production construct

![Figure 2.](https://static.igem.wiki/teams/4137/wiki/production-contruct.png)

**Figure 2.** mleR production construct: BBa\_K4137008, nickname mleR.

Here, we also used a T7 promoter, T7 terminator, and strong RBS to ensure a high, constitutive level of mleR production; when mleR binds with malate, it is activated and thus able to perform its function of transcriptional activation.

b. ccdA-ccdB toxin antitoxin constructs

![Figure 3.](https://static.igem.wiki/teams/4137/wiki/toxin-anti-toxin.png)

**Figure 3.** CcdA-production construct: BBa\_K4137012, nickname CcdA

![Figure 4.](https://static.igem.wiki/teams/4137/wiki/bruh.png)

**Figure 4.** CcdB-production construct: BBa\_K4137013, nickname CcdB

The promoter for CcdA was sourced from mleS, a protein governed by mleR in the mle operon. This regulatory site, nicknamed pmleS, promotes transcription only when bound by activated mleR; by using it as the CcdA-production construct’s promoter region, we make CcdA production inducible with malate. This allows us to achieve malate-controlled production of the antitoxin and thus enforce the permissiveness of the rhizosphere.

On the other hand, ccdB toxin is constitutively synthesized using the pLac promoter. The constitutive expression here enforces the nonpermissiveness of malate-absent environments. We added purification tags to our protein sequences for downstream purification and quantification purposes.

By designing our constructs with biobrick restriction sites bracketing the genetic components, we’re able to modularize each construct to allow for a variety of plasmid combinations; this is explained further in **Cloning Plans**. This design allows us to carry out different functional tests and verify our theory separately. For example, we could test the toxin-antitoxin theory by cloning ccdA and ccdB together in the same plasmid to prove whether both proteins bind and inhibit the toxicity of ccdB. Moreover, we can compare the results of ccdA production with and without the presence of mleR gene, which would confirm our theory of how mleR regulates the expression of ccdA.

### Cloning Plans

In order to physically create our engineered systems, we will do multiple cloning cycles to create plasmids with different combinations of our designed constructs; per biobrick assembly standard, we will be using the backbone pSB1C3. However, because of delays in receiving the distribution kit, we extracted pSB1C3 from a previous iGEM team’s plasmids (pSB1C3-RFP (BBa\_J04450) and pSB1C3-pSTE12-18) by removing RFP and pSTE12-18.

![Figure 5.](https://static.igem.wiki/teams/4137/wiki/home-page-diagrams/image1.png)

**Figure 5.** Diagram of the various plasmids we will be constructing, each containing a different combination of our designed construct.

To assemble the constructs into our desired plasmids, we will be using the EcoRI(E), XbaI(X), and SpeI(S) restriction sites in conjunction with standard cloning protocol to allow for multiple construct insertions and thus complex combinations of our designed constructs.

To insert one of our constructs into pSB1C3, we’ll cut the E and S sites for both the insert and the vector. To add a second insert into pSB1C3, we’ll cut the E and X sites, as this allows us to preserve our previously inserted construct while attaching a new section in. Using this method, we’ll construct three plasmids with singular inserts (pSB1C3::CcdA, pSB1C3::CcdB, pSB1C3::mleR) and two plasmids with dual inserts (pSB1C3::CcdB::CcdA, pSB1C3::CcdA::mleR) for the experimentation on the Toxin-Antitoxin system.

For cloning results, visit the [Engineering page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D).

## Protein Expression

### Expression of ccdA-ccdB Antitoxin System

To implement our bacteria into field conditions, we mustn't neglect the need for strong biocontainment. Our intention for the kill-switch system is to prevent the unexpected effects that the bacteria may cause, and to limit the bacteria’s influence within the banana plantation. As such, we must extensively test the constructs of this system via expression to ensure that they perform their intended function precisely.

![Figure 6.](https://static.igem.wiki/teams/4137/wiki/wow.png)

**Figure 6.** A flowchart Overview of the Toxin-Antitoxin System.

The goal of experimentation for this system is then to characterize the CcdA, CcdB, and mleR constructs to determine the holistic behavior of the rudimentary kill-switch using protein expression; from this round of characterization we can revise the construct designs to choose different promoters, RBS’s, and terminators to fine-tune the system to behave as we’d like, i.e. CcdB basal expression is higher than CcdA, mleR induction being sufficient to overcome CcdB, etc.

To examine whether our constructs built during the cloning cycles perform its intended function, we’ll verify protein expression via SDS-PAGE. If time and equipment permits, we will perform purification and quantification to collect data of protein production over time, which will aid Drylab in constructing the Toxin-Antitoxin model by providing data points for kinetic constant calculations.

For results, visit the [Engineering page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D).

### Chitinase Production

The core of our experiment is to confront FOC by degrading the cell wall with the use of Chitinase which will be secreted by our designed bacteria. In order for us to confirm that chitinase is verily secreted, we’ll transform our designed chitinase construct into DH5𝜶 E. Coli cells and grow them overnight. Part of the overnight culture would be used as the starter culture and its concentration would be verified consistently until it reaches log phase. Same as the toxin-antitoxin system, we’ll harvest and lyse the cell and use SDS-Page to verify the size of the secreted protein.

Testing secretion is not enough to prove our theory, hence we’ll investigate through assays to examine our constructs' effect on FOC. The Bradford Assay would be essential for us to do quantitative tests in the future with the respective on chitinase concentration versus FOC activity. We can also use it to determine the relation on how the secretion of chitinase is affected when we change temperature and other environmental conditions.

In addition to the level of secretion, we also wanted to determine the effectiveness of our transformed bacteria. The Co-culture Assay serves this purpose in which we can examine FOC activity by comparing how it grows when surrounded by the designed bacteria. It can also be used to show differences in the measure of time. We expect to see that the co-culture plate containing our bacteria would be able to inhibit fungal activity longer than plates where Chitinase were added instead.

For results, visit the [Engineering page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D).

## Functional Assays

### Chitinase Secretion

Since our bacteria is meant to combat FOC, a co-culture assay seems the natural way to go for a proof of concept. To demonstrate and prove the substantial inhibitory effects of our engineered bacteria against FOC is the first and most crucial step to proving the viability of our project; though the secreted chitinase Chi18h8 is proven to have said inhibitory effects against FOC, whether or not this property persists when implemented in B. subtilis remains to be seen. Before that, we must first verify protein production in E. Coli BL21.

Protein production verification for BL21 would be done by running the Bradford assay and SDS-PAGE on the supernatant of the BL21 pSB1c3::Chi18h8 LB incubation mixture and lysed BL21 cell contents.

Division of protein production verification into these two categories allows us to verify extracellular secretion of chitinase and also quantify the inefficiencies of secretion; we could then use this data to improve our protein designs to improve secretion by reducing inclusion bodies.

For results, visit the [Proof of Concept page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27proof-of-concept%27%29%20%7D%7D).

### Co-culture/Chitinase Activity Assay

We plan on utilizing a co-culture antagonism assay to verify the function of the secreted chitinase. We will use an experimental group of yeast co-cultured with our bacteria to verify that the fusion protein encoded by BBa\_K4137009 maintains the same functionalities as chi18h8; though yeast is a unicellular fungus, it nevertheless possesses the chitin cell wall targeted by chitinase. Thus, we will look for significant increases in growth inhibition in the yeast-BL21 pSB1c3::chi18h8 group as compared to the yeast-BL21 pSB1c3 control group as proof of correct function.

![Figure 7.](https://static.igem.wiki/teams/4137/wiki/pancake.png)

**Figure 7.** A diagram of the concept of our co-culture assay.

For results, visit the [Proof of Concept page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27proof-of-concept%27%29%20%7D%7D).

### CcdA Induction Expression

Due to our choice of malate concentration as the environmental factor for biocontainment, we performed intensive literature research to find transcriptional regulators that reacted to malate concentration. From a preliminary round of plausible candidate selection, we found CcpA, a malate-dependent transcriptional repressor, and mleR, a malate-dependent transcriptional activator. We couldn’t find relevant gene sequences for CcpA, so we went with mleR and adapted our Toxin-Antitoxin system to this activator. To find the regulatory region that mleR was responsible for, we looked for relevant literature on mleR’s function as a malolactic enzyme regulator and found the mleS promoter(pmleS), which should theoretically be regulated by mleR.

Though pmleS and mleR come from the same organism, because the literature source for the mleR transcriptional activator and the regulatory region supposedly recognized by mleR are different, we plan to perform protein expression of CcdA alongside mleR in various malate induction concentration trial groups to ensure that our transcriptional activator truly performs the function that it theoretically possesses. Through additional research for the mleR regulon and subsequent alignment analysis performed with Benchling, we verified the existence of a highly similar sequence in pmleS - a promising sign for our system; further research yielded a paper for an unrelated field using pmleS to achieve significant induction of luciferase production. As for the induction expression test, a control group of pSB1c3::CcdA-6xHis will be used to determine the basal production of the protein.

Beyond simply ensuring function, we plan to further characterize pmleS, as we see it to be a promoter with significant potential value in agriculture-related applications of synthetic biology - an inducible promoter responsive to a chemical present in root exudates has myriads of possible use cases, and will certainly aid the pursuit of future iGEM teams for sustainable agriculture protected from disease.

For results, visit the [Proof of Concept](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27proof-of-concept%27%29%20%7D%7D).

## References

Hjort, K., Presti, I., Elväng, A., Marinelli, F., & Sjöling, S. (2013). Bacterial chitinase with phytopathogen control capacity from suppressive soil revealed by functional metagenomics. Applied Microbiology and Biotechnology, 98(6), 2819–2828.
[Link to Source](https://doi.org/10.1007/s00253-013-5287-x)

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