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Growth curves under L-arabinose gradients induction

In order to test if the basic genome-editing part---λ-Red system works, we designed several different knock-in site pairs on E.coli chromosome and utilize antibiotic resistance gene to target them.

Firstly, from a large number of paper collection, we summarized aλ-Red protocol which tells that recombination bacteria cell preparation requires L-arabinose induction, and L-arabinose appears to inhibit cell growth due to cytotoxicity ofλ-Red proteins. Therefore, as to learn about cell growth circumstances under L-arabinose induction, and test ifλ-Red proteins get expressed on some level, we made a growth curve of gradient arabinose induced cell growth as follows. Through the curve, we can easily find that with the increase of arabinose induced concentration, cell grows slower and slower, implying the inhibition effect got strengthened.

Figure 1.The growth curves under L-arabinose gradients induction. Inhibition effect got strengthened with the increase of arabinose induced concentration.

Homologous sites choose

hen we sought to find cause our “socket.coli” needs a target site to be explored as a knock-in platform, which means all of our chromosome circuits should be inserted into such sites. Confronted with as long as 4.6Mbp E.coli genome, several criteria of our homologous site pair are supposed to be considered. Firstly ,it is not less than 40bp and more than 70bp, in that too short a site is not sufficient to implementλ-Red-mediated recombination, and too long is not efficient to add these homology sites to each ends of targeting fragments. Secondly, since the targeting fragment is modified via PCR, the secondary structure of both site pairs shouldn’t be too awful and most desirably meet the criteria of designing PCR primers. In addition, both sites are not on essential genes. Last but not the least, the region flanked by those two sites should be as suited as possible to insert 1~3 kb targeting fragment, for all of our targeting fragments vary from 1kb to 3kb.

Corresponding to the criteria mentioned before, we chose two different homologous site pairs both of which lies on the lac operon gene, which doesn’t affect the viability of bacteria cells at all. As to deal with targeting fragments of different length, two site pairs are chosen to be the socket target site. The two pairs are named respectively as HL/HR and LacHL/LacHR. Of the two, HL/HR is selected as a target for antibiotic resistant gene knock-in validation, and LacHL/LacHR is mainly used to knock in the chromosome circuit part.

Recombination towards HL/HR site pair

Through recombination towards HL/HR site pair , we demonstrate the basic knock-in function ofλ-Red system. The targeting fragments include two kinds of antibiotic resistance genes, kanamycin-resistant gene and tetracycline-resistant gene. Both antibiotic resistant gene are added HL/HR sites by two rounds of PCR.

The positive recombinational cells grow in the corresponding antibiotic LB plate followed by electrically mediated targeting fragment introduction. Using HL/HR lateral detect primer pairs, negative control gets a fragment of the length of 1767bp; on the contrary,if targeting fragment recombines towards the chromosome successfully, a band as long as 1308 bp will appear, which from the electrophoresis gel image, expected results shows up.<p>

Figure 2.Kanamycin-resistant gene mediated knock-in confirmation. Lane 1~4 represents different positive colonies. Bands of 1050bp appearing shows that recombination occurs. Lane 5 and 6 are negative controls to show the original length (1527bp) between HL/HR.

User Reviews

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••• Dharanish

Protein was expressed and visualised on SDS PAGE but could not recombine plasmids with it.

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•••• WHU-China 2023

1. Cas9 (BBa_K1218011) combined with Lambda-Red recombinases (BBa_K1433005) can achieve high genome editing efficiency in E. coli. <p>Group: WHU-China 2023 A research article indicated that Cas9 (BBa_K1218011) combined with Lambda-Red recombinases (BBa_K1433005) can achieve high editing efficiency in E. coli MG1655. The author reported 100% genome editing efficiency from randomly selected colonies(Fig1).[1] Figure 1: poxb gene knockout with 513 bp deletion: lanes 1,2 and 8 were control group samples, and others were experimental group samples. The Colony PCR product of the edited poxb gene was 1008 bp, and the original was 1521 bp. We conducted ladder experiments on the arabinose induction time to figure out the optimal duration of induced editing. As it is said that the induction should last at least for 6h[1], ladder induction was set up from 6h to 30h. Although double bents always existed, which means that bacteria are not completely edited in this colony (Fig2), 24h was considered as the optimal induction time with minimal double bents and almost 100% editing efficiency (Fig3). Figure 2: AGE results of genome editing Figure 3: Results of time ladder induction Interestingly, we found that this system has higher genome editing rates in DH5-alpha than in MG1655. However, as it’s not the main part of our experiments and the lack of time, we didn’t make deeper investigations. 2. Cas9 combined with Lambda-Red recombinases can achieve plasmid gene editing in E. coli. We confirmed that Cas9 (BBa_K1218011) combined with Lambda-Red recombinases (BBa_K1433005) can achieve plasmid gene editing. We added an N20s sequence and a batch of gRNAs targeting it into another plasmid (Fig4a). After co-transformation, we induced gene knockout for 24 hours by arabinose. It shows that many of the gRNAs (for example, NO. 11, 13, 14, 15) successfully targeted and deleted the N20s sequence (Fig4b). Figure 4a: The design of plasmid N20s gene knockout Figure 4b: The results of plasmid N20s gene knockout 3. It would be advisable to place Cas9 & Lambda-Red under an inducible promoter. The author of the research article informed us that constitutive gRNA expression has minimal impact on bacteria. However, it would be prudent to position Cas9 under an inducible promoter, such as araBAD. Cas9 can exhibit toxicity to bacteria, a phenomenon we observed in our experiments. When Cas9 is expressed through arabinose induction, the rate of culturing noticeably decreases compared to the previous conditions. Reference [1] Zhao, D., Yuan, S., Xiong, B. et al. Development of a fast and easy method for Escherichia coli genome editing with CRISPR/Cas9. Microb Cell Fact 15, 205 (2016).

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