Part:BBa_K4849032:Design
dapA - aspartokinase
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 64
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
Cyanogate
Level 1 plasmids, plCH47732, were designed using the modular platform built on assembly library of Plant Golden Gate MoClo, namely CyanoGate by Vasudevan et al. (2019) [6]. This platform provided standardised parts accordant to Cyanobacteria. In order to proceed with CyanoGate for this project, the level 0 part dapA was checked for the absence of BsaI and BpiI cut sites. As dapA had no BsaI and BpiI cut sites, it did not require domestication and could proceed with the assemblies. CyanoGate level 1 backbone primers of 50 bp were used for colony PCR for dapA.
Gene Target Amplification
From the E. coli MG1655 plate, a whole-cell PCR was performed with OneTaq® polymerase to confirm the presence of dapA
Figure 3: Whole-cell PCR to confirm the presence of gene targets in E. coli MG1655 using OneTaq® DNA Polymerase
Level 0 Transformation
Figure 4: Level 0 colonies grown overnight on spectinomycin resistance plates using a 2:1 insert:vector ratio for assembly before transformation. A: hisG, B: lysC,
C: dapA, D: Control without gene insert, E: Control without BpiI, F: Control without T4 DNA ligase.
Figure 5: Gel electrophoresis results from colony PCR for confirmation of the gene inserts hisG and dapA following extensive troubleshooting strategies. Yellow arrows point to the two colonies that were progressed to Level 1 assembly and transformation.. Note that positive and negative PCR controls were in the upper column of the same gel and did verify the PCR reaction to be working
Figure 6a: Expected band sizes from single restriction digestions run in silico with BsaI and NheI/EagI for dapA. One cut site was expected from NheI/EagI to linearise the plasmid, and two cut sites were expected from BsaI.
Figure 6b: Expected band sizes from single restriction digestions run in silico with BsaI and NheI/EagI for dapA. One cut site was expected from NheI/EagI to linearise the plasmid, and two cut sites were expected from BsaI.
Level 1 Colony PCR results
Figure 7: Colony PCR to confirm the presence of dapA for Level 1 transformation. Yellow arrows point to the two colonies that would be progressed to Level T assembly and transformation
Ninhydrin Protocol
Here's a protocol for performing the ninhydrin test on a live overnight culture of Escherichia coli (E. coli) to determine amino acid expression:
Materials Needed:
1. Overnight culture of E. coli
2. Ninhydrin solution (0.2% in acetone or ethanol)
3. 2M Sodium acetate buffer (pH 6.0-6.5)
4. Test tubes
5. Water bath or heat block
6. Pipettes
7. Microcentrifuge (optional)
8. Spectrophotometer (optional)
Protocol:
1. Prepare the Ninhydrin Solution:
- Prepare a fresh ninhydrin solution by dissolving 0.2 g of ninhydrin in 100 ml of acetone or ethanol. Mix well until the ninhydrin is completely dissolved.
2. Prepare Sodium Acetate Buffer:
- Prepare a 2M sodium acetate buffer solution at the desired pH (usually around 6.0-6.5). Adjust the pH using acetic acid or sodium hydroxide as needed.
3. Harvest Cells (Optional):
- If necessary, harvest the E. coli cells from the overnight culture by centrifugation. Wash the cells with a suitable buffer (e.g., phosphate-buffered saline) and resuspend them in a small volume of the sodium acetate buffer.
4. Reaction Setup:
- Label test tubes for each sample or standard you will be testing.
- To each test tube, add:
- 1-2 ml of the sodium acetate buffer (pH 6.0-6.5)
- 100-200 µl of the E. coli culture (or equivalent volume of harvested cells)
- If using standards, prepare a standard containing known concentrations of amino acids in the same buffer.
5. Add Ninhydrin Solution:
- Add 1-2 ml of the prepared ninhydrin solution to each test tube containing the samples and standards.
6. Heating:
- Place the test tubes in a water bath or heat block preheated to around 80-100°C.
- Incubate the tubes for about 10-20 minutes. The exact incubation time may vary based on the temperature and the specific amino acids being tested.
7. Colour Development:
- After incubation, remove the tubes from the water bath and allow them to cool to room temperature.
8. Measurement:
- Measure the colour development in each tube using a spectrophotometer at an appropriate wavelength (usually around 570 nm). Use the buffer with ninhydrin as a blank.
9. Data Analysis:
- Compare the absorbance readings of the samples. The intensity of colour development indicates the presence and quantity of amino acids in the samples.
Note: This protocol provides a basic framework for performing the ninhydrin test on live E. coli cultures to determine amino acid expression. The exact conditions and steps may need to be optimized based on the specific requirements of your experiment and the amino acids you are interested in. Always ensure proper safety precautions and handle chemicals and cultures with care.
Source
Escherichia coli str. K-12 substr. MG1655 (NCBI:txid511145)
References
[1] Toney, M.D. (2014) ‘Aspartate aminotransferase: an old dog teaches new tricks’, Archives of biochemistry and biophysics, 54: 119–127
[2] Nærdal, I. et al. (2011) ‘Analysis and manipulation of aspartate pathway genes for L-lysine overproduction from methanol by Bacillus methanolicus’, Applied and environmental microbiology 17: 6020–6026
[3] Laber, B., F. X. Gomis-Rüth, M. J. Romão, and R. Huber. 1992. “Escherichia Coli Dihydrodipicolinate Synthase. Identification of the Active Site and Crystallization.” Biochemical Journal 288: 691–95
[4] Acord, John, and Millicent Masters. 2004. “Expression from the Escherichia Coli dapA Promoter Is Regulated by Intracellular Levels of Diaminopimelic Acid.” FEMS Microbiology Letters 235: 131–37
[5] Karsten WE. Dihydrodipicolinate synthase from Escherichia coli: pH dependent changes in the kinetic mechanism and kinetic mechanism of allosteric inhibition by L-lysine. Biochemistry. 1997 Feb 18;36(7):1730-9. doi: 10.1021/bi962264x. PMID: 9048556.
[6] Vasudevan, R. et al. (2019) ‘CyanoGate: A Modular Cloning Suite for Engineering Cyanobacteria Based on the Plant MoClo Syntax’, Plant physiology 39–55
[7] Verrastro, I. et al. (2015) ‘Mass spectrometry-based methods for identifying oxidized proteins in disease: advances and challenges’, Biomolecules, 2: 378–411