Team:Manchester/Notebook
Lab Book
So much... So much happened here. And so much is about to.- Dr. Walter Bishop
FRINGE
🍀 Lab Plan 🍀
Polyphosphate Kinase Fusion Protein Kinetics
Basic aim: Assess impact localisation tag has on kinetics
Introduction/background
Our project initially aimed to screen a selection of bacterial polyphosphate kinases to select one with the greatest forward rate of reaction (polyphosphate formation). Due to time constrain, we have decided to focus on a single polyphosphate kinase that we identified in the Brenda database. It’s from Corynebacterium glutamicum, a bacteria used for industrial production of amino acids. The bacteria was discovered to accumulate high concentrations of polyphosphate (600mM, 37% cell volume), which was largely attributed to a class II Polyphosphate kinase. This PPK (encoded by NCgl2620) has a Kcat value of 74 for ATP ➝ Poly P under the experimental conditions used. It is also fully soluble, unlike the E. coli PPK which was encapsulated within a Pdu microcompartment by Warren et al. earlier this year. We hypothesise that solubility may enhance efficiency of encapsulation.
At this stage we will only be trialing one localisation tag (PduD1-20) - we will characterise the other tags using mCherry and fluorescence microscopy as an indicator for incorporation into BMC.
We were planning on ordering these sequences as Gblocks and simultaneously cloning them into both the submission vector and the expression vector pNIC-bsa28 in frame with a T7 promoter. Then we can over-express these fusion proteins in BL21 (DE3) cells and purify them using a nickel affinity resin column.
After expression and purification, the enzymes constructs will be characterised via Michaelis-Menten kinetics using the Promega ADP-Glo TM Kinase Assay, a well-plate based assay that is suitable for ‘virtually any kinase’. Technical support from Promega confirmed that the kit should work on a polyphosphate kinase. The readout for the test is based on luciferase. We managed to secure a sponsorship of 400 assays kit from Promega.
Kinetics data generated from these assays will allow us to assess any impact the localisation tag or mCherry tag has on enzyme activity. Results from this will aid how we build the construct to target the enzyme into the BMC at a later stage.
Experimental Plan
1. Design and order the following constructs with EcoRI, XbaI prefix, SpeI, PstI and XhoI suffix (XhoI will be used to go into pNIC28).
a. (N)tag_mCherry_PPK_His6
b. (N)tag_PPK_mCherry_His6
c. PPK_His6
2. Resuspend powder gBlocks (1000 ng) in 20 uL EB buffer (miniprep kit).
Clone PPK gblocks into pNIC28 (PPKs in pNIC28) and pSB1C3 (PPKs in pSB1C3)
3. Restriction digest pSB1C3 with EcoRI and PstI, pNIC28 with XbaI and XhoI and the PPK constructs (some for pSB1C3, some for pNIC28).
4. Perform ligation reaction 3 : 1 (~50ng vector).
5. Transform into DH5a cells.
6. Overnight cultures.
7. Miniprep plasmid.
8. Restriction digest some of the ligated plasmid and run agarose gel to confirm insert (Table 1).
| Construct | Digest with | Expected fragment size (bp) |
|---|---|---|
| pSB1C3_PPK_His | EcoRI + PstI | 2029 + 1121 |
| pSB1C3_PduD_mCherry_PPK_His | PstI + HindIII | 2596 + 1337 |
| pSB1C3_PduD_PPK_mCherry_His | EcoRI + HindIII | 2439 + 1494 |
| pNIC28_PPK_His | XbaI + XhoI | 5192 + 1111 |
| pNIC28_PduD_mCherry_PPK_His | XbaI + XhoI | 5192 + 1894 |
| pNIC28_PduD_PPK_mCherry_His | XbaI + XhoI | 5192 + 1894 |
9. Send all constructs for sequencing. (Sequencing primers)
10. Submit pSB1C3 constructs as new BioBricks.
11. Transform pNIC28 constructs into BL21(DE3) cells for expression.
12. Colony PCR and agarose gel - to confirm ligated plasmid is present.
13. Grow each of the confirmed transformants in TB.
14. Purify protein using nickel resin.
15. Measure specific activity of enzyme using ADPGlo Kinase Assay kit.
16. Determine kinetics with respect to PO4-.
17. Upload characterization data for submitted parts.
18. Attempt to model phosphate accumulation in E coli using PPK data.
Eut Microcompartment
Basic aim: Form a Eut BMC
Bigger aim: Optimise BMC formation
Introduction/background
We initially considered using a Pdu microcompartment to package our enzyme, as it is well characterised in literature. However, after consulting our supervisors and PIs, we have decided to use Eut microcompartments for our experiments. This particular microcompartment functions as a site where ethanolamine is cleaved into ammonia and acetaldehyde in bacteria such as Citrobacter and Salmonella. This is achieved through the action of ethanolamine-ammonia lyase, which is localised inside the microcompartment.
Localisation of proteins like the aforementioned enzyme requires the presence of a Eut signal sequence/ tags - a short sequence of 18-20 amino acids that is located at the N-terminal of the protein of interest. From literature and through consultation, we have found that EutC1-19, EutD1-20, and EutE1-20 seem to be the most well characterised of these sequences. Additionally, due to the relatedness of Eut and Pdu, Pdu signal sequences PduD1-20 and PduP1-18 have also been found to be able to localize proteins inside Eut microcompartments.
We want to build the following circuits to express the Eut proteins S, M, N, L, K under the control of inducible promoters. The operon has been split into 3 sections: each part has been designed and is ready to order as gBLocks (Table 2).
| Part | Details | Length (bp) |
|---|---|---|
| lacUV5_EutS | IPTG inducing lacUV5 promoter causing EutS synthesis. N term His tag on EutS. | 1983 |
| tetA_EutMN | Tetracyclin inducing TetA promoter causing GFP-EutM and EutN synthesis. N term His tag on GFP-EutM; C term FLAG tag on EutN. | 2452 |
| araBAD_EutLK | Arabinose inducing araBAD promoter causing EutL and EutK synthesis. N term His tag on EutL; C term FLAG tag on EutK. | 2740 |
| Eut protein | Length (a.a.) | Size (kDa) | Tags | Size with tags (kDa) |
|---|---|---|---|---|
| S | 111 | 11.6 | NTerm His6 | 12.70 |
| M | 97 | 9.4 | NTerm His6 + GFP | 40.12 |
| N | 95 | 10.0 | CTerm FLAG | 11.20 |
| L | 219 | 22.8 | NTerm His6 | 23.92 |
| K | 166 | 17.9 | CTerm FLAG | 19.10 |
Experimental Plan
1. Order 3 Eut parts as gBlocks from IDT.
Clone each Eut part into pSB1C3 and pSB4A5
2. Restriction digest pSB1C3, pSB4A5, Lac_EutS, Tet_EutMN, and ara_EutLK with EcoRI and PstI.
3. Perform the 6 ligation reactions (+ 2 controls).
4. Transform into DH5a cells.
5. Overnight cultures.
6. Miniprep plasmid.
7. Restriction digest some of the ligated plasmid and run agarose gel to confirm insert (Table 4).
| Construct | Digest with | Expected fragment size (bp) |
|---|---|---|
| pSB1C3_lac_EutS | XhoI | 1700 + 1405 + 892 |
| pSB1C3_tetR_EutMN | NcoI | 2655 + 1808 |
| pSB1C3_araBad_EutLK | BamHI | 3830 + 925 |
| pSB4A5_lac_EutS | EcoRI + XhoI | 4041 + 1281 |
| pSB4A5_tetR_EutMN | EcoRI + NcoI | 4729 + 1059 |
| pSB4A5_araBad_EutLK | BamHI | 5155 + 925 |
8. Send all constructs for sequencing (Sequencing primers).
9. Submit pSB1C3 constructs as new BioBricks.
10. Transform pSB1C3 Eut constructs into DH5a cells for expression.
11. Colony PCR and agarose gel - to confirm ligated plasmid is present.
12. Overnight cultures of each of the confirmed transformants in TB.
13. Induce pSB4A5 construct expression of each construct.
14. Check pSB4A5 construct expression by western blot.
Assemble the Eut operon in pSB1C3 using biobrick cloning sites
15. Restriction digest pSB1C3_Lac_EutS with SpeI + PstI and pSB1C3_TetR_EutMN with XbaI + PstI.
16. Ligation reaction = to form pSB1C3_Lac_EutS_TetR_EutMN.
17. Transform into DH5a cells.
18. Colony PCR and agarose gel.
19. Overnight culture.
20. Miniprep and run agarose gel.
21. Gel extraction of pSB1C3_Lac_EutS_TetR_EutMN.
22. Restriction digest with XhoI + NcoI and run agarose gel to confirm successful insert (Table 3).
23. Restriction digest pSB1C3_Lac_EutS_TetR_EutMN with SpeI + PstI and pSB1C3_araBad_EutLK with XbaI + PstI.
24. Ligation reaction = to form pSB1C3_Lac_EutS_TetR_EutMN_araBad_EutLK.
25. Transform into DH5a cells.
26. Colony PCR and agarose gel.
27. Overnight culture.
28. Miniprep and run agarose gel.
29. Gel extraction of pSB1C3_Lac_EutS_TetR_EutMN_araBad_EutLK.
30. Restriction digest with XhoI + NcoI + BamHI to confirm successful insert (Table 5).
31. Send for sequencing (Sequencing primers).
| Construct | Digest with | Expected fragment size (bp) |
|---|---|---|
| pSB1C3_Lac_EutS_TetR_EutMN | XhoI + NcoI | 2388 + 1713 + 1405 + 625 + 267 |
| pSB1C3_Lac_EutS_TetR_EutMN_araBad_EutLK | XhoI + NcoI + BamHI | 2602 + 1713 + 1554 + 1405 + 925 + 625 + 267 |
| pSB4A5_Lac_EutS_TetR_EutMN | XhoI + NcoI | 6010 + 1713 |
| pSB4A5_Lac_EutS_TetR_EutMN_araBad_EutLK | XhoI + NcoI + BamHI | 5176 + 2602 + 1713 + 925 |
32. Submit pSB1C3_Lac_EutS_TetR_EutMN_araBad_EutLK as BioBrick.
Expression of Eut operon
33. PCR the Eut operon out of pSB1C3 (VR, VF2).
34. Restriction digest Eut operon and pSB4A5 with EcoRI + PstI.
35. Ligation reaction = to form pSB4A5_LacEutS_TetEutMN_araEutLK.
36. Transform into DH5a cells.
37. Colony PCR and agarose gel.
38. Overnight culture.
39. Miniprep plasmid.
40. Restriction digest with XhoI + NcoI + BamHI and run agarose gel to confirm insert.
41. Send for sequencing to ensure no mutation.
42. Grow each of the confirmed transformants in TB.
43. Induce pSB4A5_Lac_EutS_TetR_EutMN_araBad_EutLK.
44. Confirm expression of all Eut proteins via western blot.
45. Fluorescence microscopy to visualise and confirm BMC formation - this would be the empty BMC.
Real Time Record of Lab Work
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