Primer Design for Cloning of L-arabinose isomerase Gene from Arthrobacter psychrolactophillus into plasmid pET28a(+)

A novel putative L-arabinose isomerase (L-AI) called ApL-AI with the accession number WP_110486392.1 was successfully retrieved from Arthrobacter psychrolactophilus B7 genome (Accession: NZ_QJVC01000021.1) through genome mining analysis. This study aimed to obtain the L-AI gene from the Arthrobacter psychrolactophilus B7 genome and clone it into the pET28a(+) plasmid. The primers pair designed in this study successfully amplified the gene using 60 °C of PCR annealing temperature and supported the gene amplicon to insert into the pET28a(+) to form plasmid pET28a(+)-ApLAI. It was proved by the appearance of a 1557-bp amplification band on the gel electrophoresis. The sequencing analysis also revealed that the gene was inserted in the correct direction, with the gene positioned after the promoter and finished with a terminator. Therefore, the plasmid can be used to express the ApL-AI gene to produce the ApL-AI enzyme for downstream analysis and further prospecting.


Introduction
D-tagatose is considered a rare sugar due to its rarity in nature.This ketohexose sugar is widely known as a low-calorie sugar since it is metabolized differently than dairy sugar and does not significantly affect blood sugar levels.It is also almost as sweet as table sugar but with considerably lesser calorie content, with only 38% of it in table sugar [1,2].Thus, it benefits people with diabetes, obesity, and cardiovascular disease [3][4][5][6].Further, it is not poisonous, non-carcinogenic, and safe for pregnant women if the consumption does not exceed more than 30 mg per day [2].Hence, the consumption of sugar has been generally recognized as safe by FDA since 2021 [3].
D-tagatose can be produced biologically through an enzymatic reaction with L-arabinose isomerase (L-AI) as a catalyzer (L-AI; EC 5.3.1.4).This method is environmentally safe since it avoids any toxic waste and considerably more effective than chemical synthesis of D-tagatose.L-AI is an enzyme that possesses an optimal activity to convert L-arabinose to L-ribulose [7].However, the enzyme also has an activity to convert D-galactose to D-Tagatose as the reaction product [8,9].The enzyme can isomerize IOP Publishing doi:10.1088/1755-1315/1324/1/012134 2 D-galactose by changing the aldehyde group in it to the ketose group to form D-Tagatose. Therefore, the enzyme has been widely studied for its potential in the industrial application of D-tagatose mass production for commercial purposes.
L-arabinose isomerase enzyme is widespread in microbes, reflecting the enzyme's importance for bacteria.It is due to the bacteria naturally using the enzyme to harvest the carbohydrates sourced from plant biomass.Previous studies indicated that some bacteria are sources of potential L-arabinose isomerase, including Acidothermus cellulolytics [10], Geobacillus (Bacillus) stearothermophilus US100 [11], Thermotoga maritima [12], Thermotoga neapolitana [13], and Bifidobacterium adolescentis [8].However, the exploration to discover the most valuable enzyme that meets industrial requirements.The discovery of new enzymes with superior traits, including high thermostability and acid resistance, is still needed to conduct.
Arthrobacter psychrolactophilus belongs to Actinobacteria, previously studied for its other sugarprocessing enzyme, D-allulose 3-epimerase [14].The enzyme proved superior for its thermal stability and catalytic activity.Further study on its genome also revealed the presence of the L-AI gene.Therefore, the L-AI from Arthrobacter psycrolactophilus can be explored as a potential enzyme in Dtagatose mass production.
This study was aimed to obtain the gene L-arabinose isomerase from the genome of Arthrobacter psycrolactophilus B7 and cloned the gene into plasmid pET28a(+) for further application such as the protein expression.In this study, the L-AI gene was successfully retrieved from the genome of Arthrobacter psycrolactophilus B7 and cloned into the plasmid pET28a(+) using primers designed and optimized principally to support homology recombination.The result was visualized and evaluated using agarose electrophoresis and sequenced to confirm the appropriate direction of the gene insert.

Gene mining from NCBI and the primer design
The gene mining was conducted on the BLAST-p web server on the NCBI website (https://blast.ncbi.nlm.nih.gov) using the sequence of L-arabinose isomerase originating from Geobacillus stearothermophilus US100 (Accession: CAI29261.1)as the query sequence following the step by Nirwantono et al. [15].The species filter was filled with Arthrobacter psycrolactophilus B7 to restrict the search to the selected species.The sequence matched with the BLAST-p search was opened, and the genome of Arthrobacter psycrolactophilus B7, which is bringing the L-AI sequence, was downloaded [16].Then, the DNA sequence encoding the L-AI enzyme was retrieved.
The primer design was based on the direction from In-Fusion® HD Cloning kit by Takara Bio (Kyoto, Japan) on the Benchling Bioinformatics platform [14].The forward primer was designed by combining 23 bp of DNA sequence from the 5'end of the L-AI gene and 17 bp from pET28a(+) plasmid along with its NdeI restriction site.On the other hand, the reverse primer was designed by combining 19 bp of DNA sequence from 3'end of the L-AI gene and 19 bp from pET28a(+) plasmid which brings the EcoRI restriction site.Both primers were terminated by cytosine.The primer helped the primers to anneal to the plasmid in gene cloning and to insert the gene into it.The insertion strategi is displayed in Figure 1.

In silico analysis of primers
The primer designed from the previous step was evaluated for its physical properties to ensure it could perform well to amplify the gene from the genomic DNA of Arthrobacter psychrolactophilus B7.The evaluation included the Tm of both the plasmid part and the gene part separately.The Tm of the plasmid was evaluated using the Benchling Bioinformatics platform by selecting the sequence needed to be evaluated to display the Tm and GC content.

The insert and pET28a(+) plasmid preparation
The gene was amplified to multiply the copy number of the gene and to add the overlapping portion of the plasmid for the recombination purpose.The amplification was carried out using the CloneAmp™ HiFi PCR Premix kit (Takara Bio; Kusatsu, Shiga) following the manual direction from the manufacturer and performed on the TaKaRa PCR Thermal Cycler Dice Touch (Takara Bio; Kusatsu, Shiga) to generate a 1506-bp fragment.The cycler setting was as follows: The PCR setting was conducted as follows: pre-denaturation at 98°C for 5 min; 30 cycles of denaturation (98°C; 5 sec), annealing (60°C; 15 sec), extension (72°C; 5 sec), and final extension (72°C; 5 min).
The result was monitored using horizontal 1.5% Agarose electrophoresis under 100 volts for 90 min.SYBR® Green I (final conc.1x) was used for DNA visualization under blue-light exposure, and the appeared band was then cut.On the other hand, the pET28a(+) was double-digested using EcoRI and NdeI (NEB; Ipswich, MA) employing CutSmart® Buffer (NEB; Ipswich, MA) using the standard protocol.The 1% agarose gel electrophoresis (100 volts for 90 mins) was used to separate the digestion products and visualized using SYBR® Green I (final conc.1x) under blue-light exposure.The DNA band appearing in the gel was cut.Then, both the insert and plasmid DNA were then purified using NucleoSpin Gel and PCR Clean-up (Takara Bio;12 Kusatsu, Shiga) utilizing the standard protocol.

The insertion of amplicon into pET28a(+) and E.coli transformation
According to manufacturer recommendations and experiment optimization, the insert DNA (gene) and the linearized plasmid DNA were mixed with the ratio 5:1 using In-Fusion® HD Cloning Kit (Takara Bio; Kusatsu, Shiga).After cloning, the recombinant plasmid pET28a(+)-LAI was transformed into Escherichia coli strain Stellar Competent cell (Takara Bio; Kusatsu, Shiga).Starting with 30 min incubation, the heat shock was performed to the plasmid-cells mixture at 42°C for precisely 45 sec, followed by chilling on ice for 2 min.The 500 µL of SOC medium was added to the mixture and incubated at 37°C for one h under 165 rpm of shaking.The transformant cells were poured on the LB-Kanamycin plate and incubated at 37°C overnight.The single-cell colony growing on the master plate was selected for subsequent steps.

The confirmation of gene cloning using PCR
The plasmid pET28a(+)-LAI was purified from the colony from the master plate using QIAprep Spin Miniprep Kit (Qiagen; Hilden, Germany) according to the standard protocol.The confirmation of the gene insertion was carried out by using PCR using similar primers and the protocol described previously.The result was visualized using agarose electrophoresis using the protocol used previously.

The confirmation of insert direction using Sanger Sequencing
The plasmid pET28a(+)-LAI was amplified with the T7 primer pair and Q5® High-Fidelity PCR kit using standard protocol to generate a 1557-bp fragment.The amplification was executed in TaKaRa PCR Thermal Cycler Dice Touch (Takara Bio; Kusatsu, Shiga) with cycler settings as follows: a cycle of pre-denaturation (98°C; 5 min); 30 cycles of denaturation (98°C; 10 sec), a cycle of annealing (55°C; 30 sec), a cycle of extension (72°C for 60 sec), and a cycle of final extension (72°C; 2 min).The PCR product was sent to Integrated DNA Technologies (IDT; The Gemini, Singapore) for sequencing.The quality control and the trimming of the sequencing result was conducted on the Benchling Bioinformatics platform, while the data analysis was performed by comparing the sequencing result with the sequence of LAI from the database.

DNA and Primary Protein Sequence from NCBI
The BLAST-p search resulted in a single sequence of LAI with 506 amino acids (Accession: WP_110486392.10)(Figure 2).The sequence was encoded by the LAI gene in the PRK02929 region with 1521 base pairs.The region was located at nucleotide 19039-20559 on the forward direction (+ strand) of the genome of Arthrobacter psychrolactophilus strain B7 (Accession number: NZ_QJVC01000021.1).According to the statistical calculation from the BLAST-p search, the query cover was 96% with the percent identity and the E-value of 55.67% and 0, subsequently.The sequence was homolog with the LAI from Geobacillus (Bacillus) stearothermophilus US100 and can potentially be explored for D-tagatose production.

The primers design and their physical characteristics
The primers were successfully designed with properties that are appropriate for cloning (Figure 3).According to the Table 1, the forward sequence (5' -CGC GCG GCA GCC ATA TGA TGT CTT CGC AGT TGA ATA ACT C -3') was 40 bp in total, with a GC content of 52.50%.The gene portion was 23 bp, while the plasmid portion was 17 bp.The GC content and the Tm of the gene portion of the forward plasmid were 39.13% and 60.5°C.On the other hand, the reverse primer (5' -GTC GAC GGA GCT CGA ATT CTTA GAA GCC CTG GGC GAG G -3') was 38 bp in total with a 19-bp of gene portion.The gene portion of the reverse primer would melt at 67.8 °C since it has about 63.16% of GC content.Overall, both primers supported the PCR process under the annealing temperature of PCR at about 60 °C and amplified the L-AI gene in Arthrobacter psychrolactophilus B7 genome.This step added the plasmid portion to the PCR product (amplicon) to lead to the insertion of the L-AI gene.Amplification of gene encoding ApL-AI.Lane M: DNA ladder (GeneRuler 1 kb DNA Ladder from Thermo Fisher Scientific); lane 1: negative control; line 2: gene encoding ApL-AI.

The plasmid topology after insertion and the sequence of recombinant protein
After the gene cloning, the topology of the plasmid was altered, in which the gene insertion changed the primer to be 1482-bp larger than the original.The amino acid sequence of the ApL-AI was also altered after being inserted into the plasmid since the plasmid added several additional elements into the sequence.The first element was His6tag which contains six subsequent histidine.His6tag would be helpful in protein purification using the HisTrap method (immobilized metal affinity chromatography) in upcoming step.His6tag could interact with the matrix in the chromatography column (IMAC chromatography column) and then be separated from the other proteins, which would flow through the column.On the other hand, the Trypsin cleavage site (sequence LVPRGS), a sequence in which trypsin would trim the protein, would provide a convenient property to separate the additional sequence from the primary sequence of the ApL-AI.Although, the trimming procedure is not mandatory and rare to conduct.At this stage, the plasmid is ready to be used for protein expression (expression of LAI enzyme).The protein expression could be conducted by transforming the plasmid into Escherichia coli strain BL-21 cells and induced by adding IPTG.The purification could be carried out using method mentioned above.The remaining plasmid also could be stored in the -70 °C for storage and future application including site-directed mutagenesis for further development of the enzyme.

Conclusion
According to the result obtained from the experiment, the primer designed in this study was well performed to clone the ApLAI gene into plasmid pET28a(+) to form plasmid pET28a(+)-ApLAI.The gene was inserted correctly downstream of the promoter in the correct direction.Thus, the plasmid can be used to express ApL-AI gene to produce ApL-AI enzyme for downstream analysis and further prospecting.

FDA
: Food and Drug Administration L-AI : L-arabinose isomerase D-Tagatose : a hexose monosaccharide often found in heated milk D-galactose : a hexose monosaccharide found in dairy milk L-ribulose : a pentose monosaccharide primarily found in hemicellulose and pectin L-ribulose : a pentose monosaccharide pET28a(+) : plasmid to clone the gene EC 5.

Figure 1 .
Figure 1.The strategy map to insert the LAI gene into pET28a(+)

Figure 5 .
Figure 5.The plasmid topology of pET28a(+)-ApLAI with the position of the recombinant ApL-AI gene shown in orange color block (A).The upstream of the LAI gene with its expression factors (B), and the downstream of the LAI gene with its stop codon and the terminator (C).The primary sequence of the recombinant ApL-AI was colored with green block, while its His6tag element and trypsin cleavage site shown in blue and red box.
from Neisseria denitrificans EcoRI : endonuclease enzyme from Escherichia coli strain R Tm : Melting temperature GC-content : the percentage of bases Guanine-Cytosine in DNA sequence compared to all bases PCR : Polymerase Chain Reaction G/C clamp : the bases Guanine or Cytosine existed at the 3'end of each primer T7 : a strain of bacteriophage infecting Escherichia coli Promoter : a point at which RNA polymerase initiates the DNA transcription Terminator : a point at which RNA polymerase finishes the DNA transcription RBS : ribosome-binding site His6tag : a sequence of amino acid composed by six subsequent histidine

Table 1 .
The characteristics of both forward primer and reverse primer including length, GC-content, and melting temperature (Tm)