Difference between revisions of "Part:BBa I757010"

Latest revision as of 14:03, 21 October 2019

b-lactamase TEM optimized

1. NgoMIV / AgeI protein fusion part
2. iGEM Team Freiburg 2007 and Jochen Hecky
3. Stabilized TEM beta-Lactamase

The sequence was based on TEM-116 (pUC lactamase) with mutations over TEM-1: V84I, A184V.

Additional mutations over TEM-116: V31A, A36L, L51I, R120G, E147G, H153R, V159T, M182T, L201P, I208M, E212K, A224V, A249V, T265M (numbering according to Ambler et al. 1991).

V31A, located in the N-terminal half of helix H1, identified in a mutant after genetic selection for exchanges compensating defects induced by removal of the first five amino acid residues of the mature TEM-1 protein (Hecky & Müller, 2005), increases intrinsic helix propensity.

A36L, located in the C-terminal half of helix H1, converts residue 36 to consensus, presumably increases hydrophobic contacts between helix h1 and underlying beta-sheet.

L51I, located shortly after strand S1, identified in a mutant after genetic selection for mutations compensating defects induced by circular permutation (Osuna et al., 2002) presumably improves contacts at domain interface

R120G, located at the N-terminus of helix H4, identified in mutants after genetic selection in the terminal truncation and circular permutation backgrounds, alleviates repulsive forces with helix macrodipole.

E147G, located in the N-terminal third of helix H6, identified in a mutant after genetic selection for exchanges compensating defects induced by removal of the first five amino acid residues of the mature protein (Hecky & Müller, 2005), converts residue 147 to consensus.

H153R, located at the C-terminus of helix H6, identified in mutants after genetic selection in the terminal truncation and circular permutation backgrounds, converts residue 153 to consensus.

V159T, located in a loop connecting helices H6 and H7, identified in a mutant after genetic selection for mutations compensating interface-disrupting mutations (Jochen Hecky, unpublished results).

M182T, located at the N-cap position of helix H8, identified as global suppressor mutation (Huang et al., 1997; Siederaki et al., 2001), dominant exchange in genetic selection for terminal truncation-suppressor mutations, converts residue 182 to consensus, mechanism unclear.

L201P, located at N-terminus of helix H9, identified in a mutant after genetic selection for mutations compensating interface-disrupting mutations (Jochen Hecky, unpublished results).

I208M, located in the C-terminal half of helix H9, identified in mutants after genetic selection for suppressor mutations in the terminal truncation and circular permutation backgrounds, presumably improves van der Waals contacts across the domain interface.

E212K, located at C-terminus of helix H9, identified in a mutant after genetic selection for mutations compensating defects induced by circular permutation, presumably creates favourable electrostatic interaction to D209.

A224V, resides on second crossover loop, identified in mutants after genetic selection for exchanges compensating defects induced by removal of the first five amino acid residues of the mature TEM-1 protein (Hecky & Müller, 2005), improves hydrophobic contacts between crossover loop, C-terminal helix H11 and the underlying beta-sheet, reinforces domain interface.

A249V, located in the C-terminal half of strand S4, chosen by visual inspection of x-ray structural model, should ideally increase packing interactions in the core.

T265M, only buried residue (< 5% accessible surface area) in the set, located at C-terminal end of strand S5, mutant identified after genetic selection for exchanges compensating defects induced by removal of the first five amino acid residues of the mature TEM-1 protein (Hecky & Müller, 2005), also found in clinically isolated extended spectrum lactamases, presumably acts by improving hydrophobic contacts between terminal helices and beta-sheet underneath.

Thessaly 2019 Characterization

Thessaly 2019 sought to characterize the coding sequence of TEM-optimized β-lactamase (BBa_I757010) under the regulation of the constituve Anderson Family promoters BBa_J23100, BBa_J23105, BBa_J23106, BBa_J23119. β-lactamase is an enzyme that hydrolyses β-lactams (e.g. ampicillin) and is naturally found in prokaryotic cells. A colorimetric assay has been developed using nitrocefin as a substrate which after hydrolysis from β-lactamase changes the reaction color, from yellow (380nm) to red (490nm).

To achieve that, the coding sequence was assembled with each promoter, a universal RBS (BBa_B0034) and a double terminator(BBa_B0015). The parts were cloned in pSB1C3 and pSB1K3 and transformed into E. coli DH5a competent cells.

In the photo below you can see the results of the primer addition using overhang PCR:

Figure 1. The results obtained after the PCR with the overhang primers for the different promoters of the Anderson family. We tested different annealing temperatures (45, 47 & 53℃) aiming for clear results. The expected band is at 1119bp and the ladder used was the 100bp DNA ladder by NEB.

For protein expression, the plasmids were transformed into E. coli BL21 (DE3) competent cells.

For the β-lactamase assay, we set up the following experimental design:

1. Grow BL21 (DE3) pre-culture overnight in 5ml LB (~16h) at a shaking incubator, 37℃ / 210rpm

2. The following morning, measure the OD600 of overnight cultures

3. Dilute all cultures to OD600¬ = 0.05 in M9 minimal medium

4. Grow cells 37℃ /210 RPM until OD600=0.4-0.6 (~2h)

5. Dilute all cells to the same OD600 (e.g. 0.4)

6. Load 160 of culture in a 96-well plate (do triplicates). Add 40 ul 0.5 uM nitrocefin for a final concentration of 100nM

7. Measure the absorbance at 490nm (for nitrocefin hydrolysis) and 600nm (for cell growth) every 30 seconds for 2 hours in a microplate reader. Shake between readings. Because plateau was reached within the first 30 minutes of the reaction, only those are depicted in the graph.

To ensure that the absorbance shown corresponds only to enzymatic activity by β-lactamase, we included 3 controls in the experiment. The first control has M9 medium only (no cells) and nitrocefin, the second has empty BL21 (DE3) cells (no plasmid) and nitrocefin, while the third has BL21 (DE3) cells containing the plasmid but not the part (empty plasmid). To obtain comparable results, we normalized all values by dividing OD490 by OD600.

The results are shown in the graph below

Figure 2. The hydrolysis of nitrocefin enabled by the expression of the β-lactamase gene, under the control of different promoters (J23100, J23105, J23106 & J23119) of the Anderson family. The substrate (nitrocefin) hydrolysis (490nm) is divided by cell growth (600nm), in order to normalize all values.

The maximum expression of β-lactamase was observed under control of the J23119 (brown line) which is the wild type promoter of the Anderson family. The expression is reduced with the J23100 and J23106 (yellow and purple line respectively), while the lowest expression levels were observed with the J23105 promoter (blue line). These results are in accordance with those from previous teams that measured fluorescence and the same pattern is observed. The controls conditions (pSB1C3 and BL21, or light purple and light blue respectively) confirm that the absorbance measured derives from β-lactamase activity only, both quantitatively and visually.

Below you can see the 96-well plate of the assay:

Figure 3.The observed color change due to the hydrolyzation of nitrocefin due to the production of β-lactamase, after a 2-hour enzymatic assay.

Note that the picture was taken after the plate-reader assay was completed and all conditions had reached a plateau, except the controls.

Sequence and Features