Part:BBa_C0160

autoinducer inactivation enzyme aiiA (no LVA)

same as Part:BBa_C0060 except no LVA tag

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 99
  
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization done by ETH Zurich 2015

Check the characterization done by ETH Zurich 2015 iGEM team in the experience page.

Functional Parameters: Austin_UTexas

BBa_C0160 parameters

Burden Imposed by this Part:

Burden Value: 0.8 ± 5.7%

Burden is the percent reduction in the growth rate of E. coli cells transformed with a plasmid containing this BioBrick (± values are 95% confidence limits). This BioBrick did not exhibit a burden that was significantly greater than zero (i.e., it appears to have little to no impact on growth). Therefore, users can depend on this part to remain stable for many bacterial cell divisions and in large culture volumes. Refer to any one of the BBa_K3174002 - BBa_K3174007 pages for more information on the methods, an explanation of the sources of burden, and other conclusions from a large-scale measurement project conducted by the 2019 Austin_UTexas team.

This functional parameter was added by the 2020 Austin_UTexas team.

Kinetic Parameters and Enzyme Activity in different conditions: WHU-China 2020

BBa_C0160 parameters

Fig1: Kinetic parameters of AHL-lactonase against different kinds of AHLs. a: The data are means from triplicate experiments. b: ND indicates not determined due to poor solubility of the substrate in phosphate buffer. c: 3-Hydroxylbutanoyl L-homoserine lactone.

Fig2: Effect of pH (A), temperature (B), and metal ions (C) on aiiA activity.

The Michaelis constant (Km) and kcat value of an enzyme have always been important as they show the efficiency of the enzyme towards its substrate. So they are frequently used to decide whether the enzyme is “good” or not according to specific situation. And what is also important is the conditions influencing the enzyme activity, such as temperature, PH, metal ions and so on. Here we add the data we obtain from literature[1] about aiiA gene, including kinetic parameters towards different substrates and its activity changes versus different conditions. Further users can conduct their experiments or models based on these data then.

Reference:

1.Wang L H, Weng L X, et al., Specificity and Enzyme Kinetics of the Quorum-quenching N-Acyl Homoserine Lactone Lactonase (AHL-lactonase) [J]. Journal of Biological Chemistry, 2004, 279(14):13645-51.

Protocols

Combined with the results of experiments this year, we propose a new procedure to characterize the enzyme activity.

1. Cut the plasmid with single enzyme

2. Use Gibson assembly kit to ligate it with the target gene (homologous arms and His tag were added when the genes were synthesized)

3. Transform the ligation products into E.coli BL21 and culture it after spread plate

4. Pick some single colonies into fresh medium

5. Use colony PCR to find the positive ones, and send samples to company to do gene sequencing

6. Amplify the bacteria in larger volume of fresh medium

7. Use IPTG to induce the expression of the enzymes

8. Collect the bacteria and extract the proteins

9. Keep some of the crude bacteria extract and use Ni resins (from commercial kit) to purify others

For the crude extract:

(1) Mix the crude extract and the AHL standard stock solutions in certain buffer (according to the optimal PH of the enzyme) and the control group is added with extract inactivated by high temperature

(2) React in optimal temperature and take one sample per 30 minutes

(3) Extract three times with at least an equal volume of acidified ethyl acetate

(4) The organic layer is separated, collected and dried using anhydrous sodium sulfate

(5) Use a rotary evaporator to remove the organic solvent in the sample under reduced pressure

(6) The residue is reconstituted in chromatographic grade methanol

(7) The sample is analyzed by HPLC to quantify the AHL concentration in the reaction system

For the purified enzymes:

(1) Analyze the protein with SDS-PAGE

(2) Measure the enzyme concentration by BCA kit

If the crude extract can efficiently degrade AHLs, then use the purified enzymes to repeat the former procedure performed on the crude extract; if not, change the conditions of enzyme expression to avoid inclusion-body form of the enzymes

For the standard curve:

(1) Dissolve certain amount of AHL dry powder in chromatographic grade methanol to create a series of AHL solutions with different concentrations

(2) Analyze them by HPLC and draw the standard curve of AHL concentration versus HPLC data.

10. Analyze the data from the experiments of purified enzymes with the help of the standard curve

11. Then use excel to draw the Lineweaver-Burk plot and obtain the Km (Michaelis constant) value of the enzymes towards this kind of AHL

12. The same operation can be done to attain different Km values towards 3-oxo-C12-HSL and C4-HSL in Pseudomonas aeruginosa.

13. Combine the data with our quorum dynamics model to observe the overall effects the enzymes have on the quorum sensing systems of P.aeruginosa

14. Decide whether the enzyme is suitable for our project

SCU-China 2023

AiiA is a lactonase from Gram-positive Bacilli sp.. It can hydrolyze and inactivate a variety of acyl homoserine lactones (AHLs), and it quenches quorum sensing by hydrolyzing the ester bonds present in various AHLs to prevent AHLs from binding to transcriptional regulators[1]. The AiiA sequences in iGEM Parts(BBa_C0060 and BBa_C0160) differ from the AiiA sequence we found in NCBI. Still, they are both derived from Bacillus and have similar AHL degradation activity. We use T7 promoter to express AiiA and IPTG induction system to simulate the characterization of proteins under induction. Finally, we performed protein functional verification. AiiA has a catalytic effect on a variety of AHLs, and it is expected that it can be evolved to optimize its function and become a broader-spectrum AHL-lactonase. Therefore, in this experiment, based on the structural characteristics of the AiiA protein, we randomly mutated its 65th amino acid (asparagine) and 195th amino acid (serine) to construct a mutant library. We then selected 2 mutants more likely to have active structures as candidates for functional verification.

AiiA has a catalytic effect on a variety of AHLs, and it is expected that it can be evolved to optimize its function and become a broader-spectrum AHL-lactonase. Therefore, in this experiment, based on the structural characteristics of the AiiA protein, we randomly mutated its 65th amino acid (asparagine) and 195th amino acid (serine) to construct a mutant library. We then selected 2 mutants more likely to have active structures as candidates for functional verification.

Biology and Usage

AiiA
Function	AHLs-degradation
Use in	Prokaryotes
Backbone	pET-28a
Derived from	Bacillus sp. A24

Design and Properties:

Since AiiA is a metalloenzyme, zinc ion coordination is necessary to maintain its activity. Following the general strategy of enzyme site-directed mutation[2], introducing a new salt bridge into the AiiA enzyme molecule is a way to improve its stability. Based on this, we randomly mutated its 65th amino acid (asparagine) and 195th amino acid (serine) to construct a mutant library. Due to the need for a salt bridge, we created a mutant library using basic amino acids and selected 5 mutants (Table 2) to build three-dimensional models.

Table 2 Mutations in AiiA and RMSD compared with WT
Amino acid mutation type	RMSD
N65K	0.195
N65R	0.195
N65H	0.195
T195K	0.195
T195R	0.195
T195H	0.195

For point mutations, the accuracy of homology modeling is already high enough. Therefore, we chose SWISS-Model to model our mutant proteins. We used PyMOL to analyze the RMSD values of the mutants and used CBdock2 software[4] to dock these five mutants with AHL molecules (C6-C12).(Table 3)

Table 3：Vina score and Cavity volume of all AiiA mutants

	Cavity volume (ų)	C6-HSL	C8-HSL	C10-HSL	C12-HSL
WT	707	-6.5	-6.7	-6.5	-6.2
N65K	923	-6.4	-6.6	-6.8	-6.8
N65R	991	-6.3	-6.5	-6.6	-6.7
N65H	923	-6.3	-6.9	-6.8	-7.1
T195K	922	-6.4	-6.6	-6.6	-6.7
T195R	924	-6.4	-6.5	-6.6	-6.6
T195H	922	-6.4	-6.6	-6.6	-6.7

Combining the RMSD values and docking results, we found that point mutations did not affect the enzyme's active center, and the binding energy of each mutant with the substrate was similar to that of the wild type. Considering that the 70th amino acid, which is close in spatial position to the 65th amino acid of AiiA, is glutamic acid, and the 236th amino acid, which is close to the 195th amino acid of AiiA, is aspartic acid, both amino acids are acidic. To form salt bridges, we initially selected N65K and T195R as mutation candidates.

The coding sequence of AiiA was connected to LacO/LacI (BBa_K1624002, BBa_K3257045) and pT7 (BBa_K4609008). IPTG was used to induce protein expression, simulating quorum sensing-induced protein expression to verify the degradation efficiency of the AiiA protein on the membrane. LacO/LacI are commonly found in the pET series plasmids. IPTG (isopropyl β-D-1-thiogalactopyranoside) is a molecular analogue of allolactose and cannot be metabolized by common laboratory chassis such as E. coli. IPTG has the same function as allolactose. Both can act as inducers and bind to the repressor in the Lac operon, thereby preventing LacI from binding to LacO upstream of pT7 and ultimately initiating the expression of AiiA. AiiA crude extract was collected and used to treat E.coli DH5α(with p15A-lux-sfGFP). Without AiiA, the LuxR secreted by J23100 in plasmid p15A (BBa_C0062) interacts with AHLs and initiates LuxP, expressing the green fluorescent protein sfGFP to emit fluorescence. When AiiA exists, AiiA degrades AHLs, LuxP cannot turn on the expression of sfGFP, and the green fluorescence weakens.

Figure.1 AiiA characterization gene circuit

Figure.2 AiiA functional verification circuit

Figure.3 pET-28a-AiiA-C-His plasmid

Figure.4 The inhibitory effect of AiiA on SRB biofilm（7h）

AiiA has good degradation effect on the biofilm of SRB. This shows that AiiA can quench quorum sensing molecules and thereby block biofilm formation.

Figure.5 Degradation effect of AiiA crude extract at different concentrations on oxo-C6-HSL

4mL of AiiA protein solution extracted from 30ml of bacterial solution, at dilution factors of 30ul/10ul/1ul (30ul in total), has a similar degradation effect on oxo-C6-HSL .

Figure.6 Degradation effects of different AHLs by AiiA crude extract

For different AHLs, after adding 30ul of undiluted enzyme, the fluorescence intensity of all treated experimental groups was lower than that of the untreated experimental group. In general, the fluorescence intensity decreased overall.

Experimental approach：

1. Express and extract proteins

(1) Transform pET-28a-AiiA-C-His into DH5α strain (K+);
(2) Pick a single colony and culture it in K+ LB liquid medium overnight at 37°C and 220rpm;
(3) Extract the plasmid, transform the plasmid into BL21 (DE3), sequence the normal bacteria, and culture it in K+ LB liquid medium at 37℃ and 220rmp overnight;
(4) Take 1mL bacterial liquid cultured overnight and add it to 50 ml (250 ml Erlenmeyer flask) of K+ LB liquid culture medium, and expand the culture medium at 37°C , 220 rpm for 4 hours until OD600 =0.6-0.8;
(5) Take 10mL of bacterial liquid and store it at 4℃ for later use. Freeze 40mL of bacterial liquid at 4℃ for 5 minutes and then add IPTG (working concentration is 1mmol/L) and induce at 28℃, 200rmp for 12h;
(6) Adjust OD600 of the induced bacterial liquid to approximately the same value. Take 10mL of the induced bacterial liquid and store it at 4°C;
(7) Take 30 mL of the induced bacterial liquid, centrifuge it at 8000 rpm, 4°C for 10 min, and take 1mL of the supernatant for SDS-PAGE verification;
(8) Resuspend the pellet in 5ml 1x PBS, centrifuge at 8000rpm, 4°C for 10 minutes;
(9) Resuspend the pellet in 4ml of bacterial protein preparation lysate(with Tris-HCl) Add 1uL DNase/RNase; dispense into 2mL centrifuge tubes; incubate at 37°C, 600rpm for 30 minutes;
(10) 30% Ultrasonic power, lyse for 10 seconds, rest for 10 seconds, a total of 10 minutes; 5 minutes interval, repeat 2-3 times;
(11) Centrifuge at 13000g, 4°C for 30 minutes, take the supernatant as crude protein solution, and store it at -20°C;
(12) In a clean bench, filter the crude protein solution with a 0.45μm filter membrane to sterilize.

2. 96-well microtiter plate assay (Crystal violet staining of biofilms)[3]

(1) Incubate the bacterial solution overnight for 12 hours until the OD600>1. Add antibiotic-free LB dilution at a ratio of 1:10. Add 125µl of the diluted bacterial solution to each well of a 96-well plate. Inoculate and incubate overnight at 37°C without shaking for 24 hours. (LB-only medium is required as a control)
[SRB bacterial film needs to be cultured in an anaerobic bag for 7 hours]
(2) Aspirate the LB, add 150ul of lysis supernatant containing induced expression protein (sterilized), and place at a constant temperature of 37℃ Celsius for 18 hours.
[Wash the 96-well plate with biofilm twice with 200ul of sterile water, add 100ul of lysis solution, scrape off the film with a pipette tip, and spread it on the plate. After 12 hours, observe the number of single colonies growing on the plate.]
[It is also feasible to directly stain and observe the growth of a certain type of bacterial film without adding lysis solution.]
(3) Aspirate the liquid, gently soak the well plate in 1L of distilled water and wash it twice. When the plate is submerged, gently wipe the surface of the plate with gloved fingers to release air bubbles and ensure that water enters. Turn the plate up side down and tap hard. Place the 96-well plate upside down on absorbent paper to remove as much water as possible.
(4) Add 200μL of 0.1% crystal violet solution (containing 5% methanol) into the well. This volume ensures that the stain covers the biofilm. Let sit for 10 minutes. Invert the plate in the waste tray and shake gently to remove the liquid.
(5) Gently soak the well plate in 1L of distilled water and wash it twice, and rub the entire surface of the plate to ensure that water enters all wells. Remove the plate from the water, invert, and shake to remove liquid. Replace with distilled water and repeat the above steps twice.
(6) Turn the plate upside down and tap hard. Place the 96-well plate upside down on absorbent paper to remove as much water as possible.
(7) Place the washed 96-well plate into the oven until the water is completely dry
(8) Add 200ul of 95% ethanol to each well and wait for 10 minutes until the crystal violet is completely dissolved.
(9) Use a microplate reader to measure the OD570nm (at least 3 repeat groups)
Note：If you’d like to count the single colonies on the plate that spread the biofilm, it is better to use resistant membrane-producing strains to avoid contamination.

3. AHLs Degradation Assay

(1) Use E.coli DH5α with the p15A-LuxR-sfGFP plasmid, set a control group, and measure the fluorescence intensity to reflect the degradation effect of the enzyme on AHLs.
(2) Cultivate the newly transformed E.coli DH5α (with p15A-lux-sfGFP) overnight for about 8 hours, and adjust the OD600 to about 0.6.
(3) Add 250ul of bacterial solution to each well; add 10ul of AHLs to the AHL-treated wells; the total volume of the protein solution is 30ul.
(4) Set the microplate reader program to measure the fluorescence and OD600 of the entire 96-well plate every 10 minutes for a total of 2 hours and 11 test values. Finally, measure the fluorescence of the odd-numbered column and the OD600 of even-numbered column, and use fluorescence intensity/OD600 to evaluate the intensity of bioluminescence.
[Note: The bacterial solution should be added last to avoid inaccuracy due to differences in operating time]

4. PCR-based Site-directed Mutagenesis Method

(1) Design primers: Select the mutation point and 15bp before it, 33 bp in total, as F-primers. Select the mutation point and 15bp after it, 33 bp in total, as R-primers.
(2) PCR(25μL in total/50μL in total); After electrophoresis and gel recovery, obtain the linearized mutant plasmids.
(3) Digest the original plasmid with DpnⅠ, and then use ligase to circularize the plasmid to obtain the mutant plasmid.

Reference

[1] Chirag Beladiya, Rajan K. Tripathy, Priyanka Bajaj, Geetika Aggarwal, Abhay H. Pande,Expression, purification and immobilization of recombinant AiiA enzyme onto magnetic nanoparticles,Protein Expression and Purification,Volume 113,2015,Pages 56-62,ISSN 1046-5928.
[2] Mabrouk SB, Aghajari N, Ali MB, Messaoud EB, Juy M, Haser R, Bejar S. Enhancement of the thermostability of the maltogenic amylase MAUS149 by Gly312Ala and Lys436Arg substitutions. Bioresource Technology, 2011, 102(2): 1740-1746.
[3] Coffey, B.M., Anderson, G.G. (2014). Biofilm Formation in the 96-Well Microtiter Plate. In: Filloux, A., Ramos, JL. (eds) Pseudomonas Methods and Protocols. Methods in Molecular Biology, vol 1149. Humana, New York, NY.