Overview

Iterative design is an approach to a cyclical process based on the principles of synthetic biology design: design, build, test, and learn. In the iGEM competition, we focused on engineering thoughts and aimed to improve the foundations and applications of our designs through the iterative process. In the process of carrying out this cycle, we strove to be close to the user needs, solve the pain point of efficiency improvement of the gene editing tools at this stage, and improve our project in both versatility and stability. Therefore, we believe that engineering success should exist in all the aspects of the project design, build, test and optimization, and this year we mainly focused on the build and optimization of the purification system.

Cycle1 galK gene inactivation

Research: Which kind of gRNA and donor DNA is a good start for the construction of genome editing method?

Our project aims to improve the gene editing efficiency by constructing a purification system (strainer). The describe of the strainer method can be found in home page. Gene inactivation is a frequently-used step in the strain construction. Thus, we use it as a good start for our method design and construction. The galK gene is one of the popular targeting gene in E. coli because the cells with the inactivation of galK gene will be observed as white colonies on the MacConkey agar. Moreover, the wild-type control will be observed as red colonies on the MacConkey agar. To this end, we designed four gRNAs (in the EC21 to EC24 plasmids) targeting on the different position of the galK gene and related donor DNAs contained stop codon (TAA). In addition, each gRNA with its related donor DNA is constructed in the same editing plasmid, which is good for genome editing using gRNA libraries.

Design: How can we screen an editing plasmid for gene inactivation that can be used for the construction and optimization of the "strainer" method?

In the first iteration of the design, we used two E. coli strains (EC85 and EC88) with the plasmid harboring lambda red system and different CRISPR proteins (Fig. 1) as host strains to test our four editing plasmids. Then, we can compare the editing efficiencies using four editing plasmids with two different Cas protein (MAD7 and AsCas12a). The editing plasmid with lowest editing efficiency can be used for the design and construction of the third plasmid of strainer system.

Build: How do we construct the strain with the galK gene inactivation?

We firstly prepared the electrocompetent E. coli MG1655 cells, and used electroporation to introduce the plasmid harboring lambda red system and different CRISPR proteins. The resulting strains are EC85 and EC88, which grow on the plates with Chloramphenicol. Then, we prepared the electrocompetent EC85 and EC88 cells for the galK gene inactivation. During the competent cell preparation, we added 0.2% arabinose as an inducer for Cas system, and culture cells using 42 degree 15 min to induce the lambda red system before chilling the cells (the step starting the competent cell preparation). Then, we transformed the four editing plasmids into EC85 and EC88 using electroporation, and cultured the cells on the MacConkey Agar with 1% galactose.

Test: How do we characterize the results of the galK gene inactivation?

The colonies observed as white color were the strains with the galK gene inactivation. The colonies observed as red color were the wild-type strains (Fig. 2). The editing efficiency was calculated using the formula as follows:

Learn: Which editing plasmid for the galK gene inactivation that can be used for the construction and optimization of the "strainer" method?

We compared the editing efficiency using different editing plasmid for the galK gene inactivation. When using the editing plasmid EC21, the editing efficiency in EC85 and EC88 are both lowest compared to other editing plasmids (Fig. 3). Thus, we use the plasmid EC21 as the template for the design and construction of the third plasmid of strainer system.

Cycle 2 Construction and validation of the "strainer"

Research: What is the principle of the "strainer" we built?

The previous studies have demonstrated that the SOS response is the first DNA repair system described in E. coli. The transcription of genes such as lexA, sula, umuD and recA are regulated by the SOS response. Thus, we can use the promoters of these genes to start the transcription of gRNAs targeting on the plasmid harboring a counter-selection markers if there is a double-stranded DNA breaks (DSBs) on the genome. When the sacB plasmid is cured by CRISPR/Cas system, the strain with successful recombineering can survival in the media with sucrose (Fig. 4). The strain without DSBs, still has the plasmid harboring sacB gene, cannot survival in the media with the sucrose. This is why we can increase the overall editing efficiency.

Design: How did we design the "strainer"?

Firstly we designed a second plasmid harboring sacB gene in the EC85 or EC88, and used Trc inducible promoter to control its expression. Then, we designed another gRNA that can be inserted in the EC21 plasmid. This gRNA target on the second plasmid harboring sacB gene, and use the promoter of genes such as lexA, sula, umuD and recA.

Build: How can we construct and validate the "strainer"?

We ordered the second plasmid harboring the sacB gene with Trc inducible promoter. Then, we prepared the electrocompetent EC85 and EC88 cells and transformed the second plasmid into EC85 and EC88. We also ordered four DNA fragments contained the same spacer targeting on the second plasmid with the promoter of lexA, sula, umuD and recA genes. Then, we constructed these new editing plasmids by Gibson assembly using EC21 as template.

We firstly prepared the electrocompetent cells for the galK gene inactivation using the strain EC85 with sacB plasmid. During the competent cell preparation, we added 0.2% arabinose as an inducer for Cas system, and culture cells using 42 degree 15 min to induce the lambda red system before chilling the cells (the step starting the competent cell preparation). Then, we transformed the new editing plasmid (LexA) with the lexA promoter into the strain EC85 with sacB plasmid using electroporation, and cultured the cells on the MacConkey Agar with 1% galactose and different concentration of sucrose.

Test: Which condition will be good for the "strainer" system?

We firstly tested the new editing plasmid LexA using lexA gene promoter in EC85 to validate the "strainer" method. At a 0.01% sucrose concentration, the highest CFU/μg and the highest editing efficiency was found in the experiment. The editing efficiency using LexA plasmid is 25% higher than the control without "strainer" system. However, the CFU/μg decreased significantly compared to the control group that did not use "strainer". In addition, we used the 0.01% sucrose to tested another three new editing plasmids. All of them showed that the editing efficiency is 25% higher than the control without "strainer" system (Fig. 5A).

Learn: What does we learn from the validation of the "strainer"?

Firstly, the "strainer" system significantly improves the gene editing efficiency compared to the control without "strainer" system. Secondly, the CFU/μg is still 96% lower than that of the control even using 0.01% sucrose (Fig. 5B). To this end, we should further improve CFU/μg by designing and modifying SacB protein to reduce its toxicity.

Cycle 3 Protein modification

Research: How to attenuate toxicity by engineering SacB protein?

The lethal mechanism of the sacB toxicity gene is that the protein expressed by the sacB gene (levansucrase) can catalyze the hydrolysis of sucrose into glucose and fructose, and polymerize fructose into levan. The large accumulation of levan in E. coli will kill the cells. The S164 forms a hydrogen bond with the nucleophilic agent D86 and the 4-OH of the fructose group, and S164 is important to ensure the position stabilization of D86 (Fig. 6). If we design some mutation in S164 site, the mutant could reduce the hydrolysis rate, and then the toxicity of SacB protein will reduced.

Design: How to design a SacB mutation that could decrease its toxicity for our "strainer" system?

The position of the carboxyl group of D86 is restricted by hydrogen bonding. We speculate that the S164T mutation with an additional -CH3 group would change the orientation of the-OH and would effectively form new hydrogen bonds. If hydrolysis rate reduce, the toxicity of SacB protein will reduced. Thus, we test S164T mutation using Molecular dynamics (MD) simulations. The results showed that This mutation breaks the delicate balance of the ternary catalytic amino acid with the ligand (Fig. 7). Therefore, we speculate that it will lead to reduced cytotoxicity. To this end, we sought to apply S164T mutation in the wet-lab experiment.

Build: How to construct the SacB_S164T mutation?

We followed the instruction of Mut Express II Fast Mutagenesis Kit V2 to design the primer. Then, we used the following PCR conditions to construct the SacB_S164T mutation. Thirdly, we followed the instruction of Mut Express II Fast Mutagenesis Kit V2 to construction the SacB_S164T mutant.

We prepared the electrocompetent cells for the galK gene inactivation using the strains EC85 and EC88 with SacB or SacB_S164T mutation. During the competent cell preparation, we added 0.2% arabinose as an inducer for Cas system, and culture cells using 42 degree 15 min to induce the lambda red system before chilling the cells (the step starting the competent cell preparation). Then, we transformed the editing plasmid (LexA) with the lexA promoter into the strain using electroporation, and cultured the cells on the MacConkey Agar with 1% galactose and different concentration of sucrose.

Test: How to evaluate SacB_S164T mutation?

The results showed that the editing efficiency of SacB_S164T mutant (in EC85) is 25% higher than the control without "strainer" system. The CFU/μg of SacB_S164T mutant (in EC85) increased 3-fold compared to the original "strainer" system while still keep high editing efficiency (Fig. 8A).

We also used the same condition to test SacB_S164T mutant in EC88. The results showed that the editing efficiency using "strainer" system with SacB_S164T mutant is 4-fold higher than the control, although the CFU/μg of SacB_S164T mutant (in EC88) is still 71% lower than the control without "strainer" system (Fig. 8B).

We also used the same condition to test SacB_S164T mutant in EC88. However, the results showed that the toxicity of the modified SacB protein was low, and the screening effect served by the lower concentration of sucrose chosen at this point was no longer obvious, so we further optimized the sucrose concentration and found that testing the mutants at 0.1% concentration conditions, the CFU/μg using the "strainer" system with the SacB_S164T mutant was 2-fold higher than the control, although the SacB_ S164T mutant (in EC88) still had a lower CFU/μg than the control without the "strainer" system.

Learn: How toxic is the SacB mutation?

These results showed that our strainer system works as a good purification system to remove the unedited cells, and SacB_S164T mutant is less toxicity than the original SacB.

Cycle 4 Insertion of the long gene fragment

Research: Why do we do the long gene fragment insertion work?

The "strainer" method improved the overall editing efficiency by removing the unedited cells. We have previously validated the feasibility of improving editing efficiency by introducing the "strainer" method for screening in the single gene editing, but we sought the "strainer" to potentially serve as a universal method for cell factory construction. Therefore, we applied the "strainer" to a large fragment gene editing process to verify the universality of the system.

Design: How to compare the efficiency improvement of the "strainer" method for the long gene fragment editing?

SS9 is a safe locus in the E.coli genome, where we can insert long gene fragment without strong effect on the growth of E.coli. Therefore, we chose this region to verify the "strainer" for long DNA fragment insertion experiment. We chose the CRISPR/Cas9 gene editing method as the control group and used CRISPR/Cas9 to construct the "strainer" method as the experimental group, because the CRISPR/Cas9 is also commonly used in the genome editing field. We introduced the isopropanol (IPA) production pathway (~5000 bp) into the strains using both methods to compare the efficiency of long gene fragment insertion.

Build: Introduction of IPA fragments into EC86 strain using two methods.

We firstly constructed EC86 strain which is MG1655 with the plasmid harboring lambda red system and Cas9 protein. Then, we introduced the plasmid with sacB_S164T into the EC86. Thirdly, we introduced the gRNA_SS9 plasmid and the genome fragment of the IPA pathway into host strain using two methods. The strains after transformation were spread on the LB plates, and then using colony PCR to verify the efficiency of long gene fragment insertion.

Test: How to know that the long gene fragment was inserted successfully?

We selected 30 colonies that were labeled and added to the reaction solution for colony PCR, and performed colony PCR under the reaction conditions shown in the table 2. Then we observed the running gel results, and a band at the 600 bp position was considered as a successful import.

Learn: What do the statistic comparisons show?

Through the three repeated experiments of the long gene fragment insertion, 30 samples were detected by colony PCR each time, and the electrophoresis results were observed to calculate the proportion of the colonies that had inserted IPA pathway gene fragments. We found that the efficiency of successfully inserting long gene fragment using the CRISPR/Cas9 method was very low, and even there were cases where the introduction was not successful at all. In contrast, the use of the "strainer" method effectively improved the editing efficiency of the long gene fragment insertion, so that the insertion efficiency is about 30%. (Fig. 9)

Cycle 5 Fermentation for IPA production

Research: What is the effect of "strainer" on IPA production fermentation after introducing point mutation?

We constructed the EC61 strain by inserting the IPA production pathway into the genome in MG1655, and then we further modified the EC61 strain by introducing the FliA_R94K mutation that confers isopropanol resistance.

Design: How to design a controlled trial?

We used the "strainer" and the original CRISPR/Cas methods for the introduction of FliA_R94K mutations into the EC61 strain. The "strainer" method had 20% higher editing efficiency than the original CRISPR/Cas method (Fig. 10A). Then we set EC61 as the control group and EC65 as the experimental group. Three shake flasks were set up in each group to culture and ferment as the parallel samples, two of which were sealed with parafilm and one with tinfoil.

Build: How should the fermentation experiments be conducted?

We added 24 mL of SD-8 medium, 1.25 mL of 400 g/L glucose, 20 μL of trace elements, and antibiotics to each shake flask (10 μL was taken to measure pH before inoculation to ensure that the pH range was around 6.0 before inoculation) in the reagent preparation phase of the shake flask fermentations. SD-8 medium (NH4Cl, 7.0g/liter; KH2PO4, 7.5g/liter; Na2HPO4, 7.5g/liter; K2SO4, 0.85g/liter; MgSO4·7H2O, 0.17g/liter; trace elements, 0.8 mL/liter; yeast extract; 10g/liter) containing 2% glucose was used for fermentations. The trace element solution contained the following (in grams per liter of 5 M HCl): FeSO4·7H2O, 40.0; MnSO4· H2O, 10.0; Al2(SO4)3, 28.3; CoCl2·6H2O, 4.0; ZnSO4·7H2O, 2.0; Na2MoO4·2H2O, 2.0; CuCl2·2H2O, 1.0; and H3BO4, 0.5. For antibiotic selection, the concentrations of antibiotics were 100 μg/mL (Ampicillin) and 34 μg/mL (Chloramphenicol). We took samples by shaking a dozen times after inoculation, centrifuged and kept the supernatant in the freezer for HPLC detection, measured pH, measured sugar concentration and recorded data. The shake flasks were incubated at 37°C and 200 rpm (after 12 h incubation, the speed could be slightly lowered at 180 rpm).

We found that the pH of EC65 decreased significantly after 12 h, so we added a small amount of 5M NaOH to keep the pH of the culture solution at about 6.0; when the result of our sugar measurement was less than 10 g/L, we added glucose to make the concentration of glucose in the shake flasks to around 20 g/L, and then shaked the flask for a dozen times after the sugar was added. Take the sample and store it in the freezer.

Test: How did we characterize the IPA production of the different strains?

We stored the samples taken during the fermentation process in the freezer. After completing the 48 h fermentation process, we used HPLC to detect glucose and IPA concentrations, and finally calculated the IPA yield and productivity of the EC61 and EC65 fermentations.

Learn: Comparison of the IPA production of the two strains.

We could see that the isopropanol titer of EC65 was 3-fold higher than that of EC61 from the result of HPLC (Fig. 10B). In the process of strain modification for IPA production, the "strainer" method could significantly accelerate the construction speed of the strains.