Experiments | Gifu

Introduction

At the beginning of the project, we planned to test ESBL (extended-spectrum beta-lactamase)-producing bacteria, which have many closely related species, as a model for drug-resistant bacteria. However, in the process of conducting the experiment, it was found that Cas9 was designed with an erroneous backbone Cas9 plasmid. Therefore, to solve this problem and quickly construct Cas9, we completed the engineering by using DNA that we had (the iGEM Kit plate).

In this experiment, we aimed to create a Cas9 plasmid that can easily insert various gRNAs. After Cas9 was created, we selected a model of drug-resistant bacteria from the iGEM Kit plate for additional experiments to bring our project closer to social implementation.

Cycle1: Creation of Compatible Cas9 Plasmid

Design

The plasmid was designed as the following plasmid map.
We constructed a Cas9 plasmid that can easily insert various gRNA.

Figure1-1. plasmid map of Cas9_Bbs1_plasmid

Build

Nine DNA fragments were introduced by Gibson Aseembly into the backbone Cas9 plasmid. The assembly kit NEbuilder was used for this assembly. All DNA fragments and backbone Cas9 plasmid were prepared from plasmids from the iGEM Kit plate.

In the area where the future gRNA will be inserted, a mCherry with a Bbs1 cleavage site was introduced. In this way, the Cas9 plasmid with gRNA can be easily completed by treatment with Bbs1. In addition, the use of a lac promoter as a promoter for the Cas9 protein prevents E. coli from being overloaded by the constant expression of the Cas9 protein.

Chloramphenicol resistance gene was introduced to allow screening in simultaneous transformation with ampicillin resistance plasmids.

Test

1.Fragment and Backbone Amplification by PCR

Since mCherry is flanked by Bbs1 site, we mutated the sequence by PCR.

Figure1-2. parts collection

2. Gibson Assembly by NEbuilder

We created the final Cas9_Bbs1_plasmid using the intermediate plasmids produced in test 2.

4. Transformation of the Plasmid

We transformed the plasmid in medium containing chloramphenicol and kept this recombinant E. coli (NEB5-alph) as glycerol stocks.

5. Shaking Culture and Purification

We picked red colonies expressing mCherry and extracted them after shaking culture.

Learn

In this cycle, we succeeded in designing a compatible Cas9 plasmid that can be easily introduced with a variety of gRNAs by enzyme digestion. However, we were unable to verify this by SDS page or DNA cleavage assay. Therefore, we could not increase our confidence in the expression and activity of Cas9.

Cycle2: Creation of Model Gene

Design

The plasmid was designed as the following plasmid map.

Figure2-1. plasmid map of Amp_GFP_plasmid

Plasmids with AmpR and GFP genes were constructed to compare E. coli growth by fluorescence. We used BBa_J428326 containing GFP as a coding sequence and BBa_J435300 containing AmpR as backbone.

Build

Transformation was performed using BBa_J428326, BBa_J435300 from 2023 iGEM kit plate. Then, plasmids were extracted from the cultured colonies to obtain two types of plasmids for use in the construction of the target plasmid. The plasmids BBa_J428326 and BBa_J435300 obtained above were ligated using Bsal and T4 DNAligase to create the target plasmids. After enzyme treatment, the colonies were cultured in Amp medium for selection.

Figure2-2. outline of experimental system

Test

1. Preparation of Plasmids

We transformed the BBa_J428326 and BBa_J435300 plasmids and purified the plasmids.

Figure2-3. parts collection

BBa_J435300 worked out well. BBa_J428326 was successfully transformed, and colony growth was observed on plates, but growth in shaking culture was poor. The latter was successfully twice, and once after three rounds of shaking culture. (1 out of 8 test tubes)

2. Plasmid Digestion and Plasmid

The purified plasmid was ligated with Bsal and T4 DNAligase to create the target plasmid.

3. Transformation of Target Plasmid

We transformed the target plasmid in Amp medium.

White colonies and green colonies were observed on the medium. The green colonies express GFP and are resistant to Amp, so they are target plasmids.

Figure2-4. Colonies expressing GFP in ampicillin medium

4. Shaking Culture

We picked green colonies and cultured them in Amp liquid medium with shaking.

3.4 was repeated with similar results and the target plasmid could not be obtained.

Learn

In this cycle, E. coli growth in shaker culture was poor when GFP was incorporated into the plasmid. We thought the expression of GFP was overloading E. coli and decided that it would be difficult to proceed with the experiment using this GFP. Therefore, we decided to use BBa_J435300 itself as a model for the drug resistance gene. In addition, the final confirmation of the E. coli growth method was changed from fluorescence measurement to colony count measurement.

Cycle3: gRNA Synthesis and introduction

Design

The DNA encoding crRNA was designed as shown in the following figure. The sequence was determined to recognize the drug resistance gene modeled here and another drug resistance gene.

Figure3-1. the crRNA sequence

The plasmid with gRNA was designed as the following plasmid map.

Figure3-2. plasmid map of Cas9_gRNA_plasmid

Build

In this cycle, we constructed a functioning Cas9 plasmid. By digesting Cas9_ mCherry_plasmid with Bbs1, gRNA can be introduced into the Compatible Cas9 Plasmid created in Cycle2.

Figure3-3. method of gRNA introduction

Test

1. Golden Gate Assembly

2. Transformation of the Plasmid

We transformed the plasmid in medium containing chloramphenicol and kept this recombinant E. coli (NEB5-alph) as glycerol stocks.

3. Shaking Culture and Plasmid Purification

We picked white colonies and shake cultured them. For further confirmation, we also checked whether the target plasmid was transformed into the bacteria after shaking culture. If the gRNA was not successfully introduced, the pellet collected by centrifugation was colored.

Figure3-4. left: case where gRNA is successfully introduced / right: case where mCherry still in place

Learn

This cycle showed successful introduction of gRNA into the compatible Cas9 Plasmid created in Cycle 1. However, the probability of success of Golden Gate Assembly in Test1 was extremely low, and most of colonies expressed mCherry in the first assembly. Therefore, in the second Golden Gate Assembly, the amount of insert (gRNA) was increased to 4 times the amount of vector (Cas9_Bbs1_plasmid), and the digestion time and ligation time were extended. In order to get closer to social implementation, we moved to the next step to carry out experiments against drug-resistant genetic strains.

Figure3-5. left: the first assembly / right: the second assembly

Check here for the trial and error journey of PAMConAligner!

１）　 A versatile genetic engineering toolkit for E. coli based on CRISPR-prime editing 　　／ Yaojun Tong, Tue S. Jørgensen, Christopher M. Whitford, Tilmann Weber 　　 & Sang Yup Lee／ PMID: 34471126／PMCID: PMC8410854／DOI: 10.1038/s41467-021-25541-3 (2021)

２） A simple and economical site-directed mutagenesis method for large plasmids by direct transformation of two overlapping PCR fragments ／ Zhibo Yang, Zan Chen ＆Yueping Zhang ／ PMID: 36398840／ DOI: 10.2144/btn-2022-0085（2022 ）