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 microcompartment.

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™ 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 microcompartment 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).

Table 1: Expected results from restriction digest of all PPK constructs.
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 ADP-Glo™ 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 bacterial microcompartment(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).

Table 2: Eut constructs.
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

Figure 1: 3 Eut operons. (From top to bottom) lacUV5_EutS, tetA_EutMN and araBAD_EutLK.

Table 3: Sizes of Eut proteins with and without tags.
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).

Table 4: Expected results from restriction digest of all Eut constructs.
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).

Table 5: Expected result from restriction digests during Eut operon assembly.
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.

Localisation Tag Characterisation

Basic aim: Encapsulate mCherry tagged with the signal sequences inside the Eut microcompartment

Introduction/background

Successful encapsulation would be justified by fluorescence microscopy using colocalisation technique, which involves the observation of the spatial overlap between two different fluorescent labels, i.e. mCherry and GFP, each having a separate emission wavelength. Microcompartments carry GFP while the localisation tags carry mCherry.

1. PCR out the RBS_tag_mCherry constructs from Ash’s plasmids (Table 6).

2. Cloning:

a. Cut all 5 tag constructs with EcoRI and PstI to clone into pSB1C3 submission vector with medium strength constitutive promoter (BioBrick: BBa_J23108).

b. Do a restriction digest. Cut the promoter vector (pSB1C3 which contains the promoter BBa_J23108) with SpeI and PstI and cut the PCR product with XbaI and PstI. Use a phosphatase to cleave the 5’ phosphate in the promoter vector.

3. Sequence and submit as new parts (or improved parts for PduD and EutC).

4. Confirm expression - agarose gel electrophoresis.

Table 6: Tag constructs (Ash’s stock).
Tag Name	Vector	Amino acid sequence
EutC(1-19)	pETDuet	MMDQKQIEEIVRSVMASMGQ
EutD(1-20)	pETDuet	MIIERCRELALRAPARVVFP
EutE(1-20)	pETDuet	MNQQDIEQVVKAVLLKMQSS
PduD(1-20)	pETDuet	MEINEKLLRQIIEDVLRDMK
PduP(1-18)	pETDuet	MNTSELETLIRTILSEQL

Selecting constitutive promoters from the registry

We have chosen medium, strong and weak constitutive promoters for expression of the tag-mcherry parts:

a. Part:BBa_J23108 - strength = 0.51 - plate 4 - well 4C

b. Part:BBa_J23104 - strength = 0.72 - plate 4 - well 17L

c. Part:BBa_J23105 - strength = 0.24 - plate 4 - well 17N

The strong and weak promoters (Part:BBa_J23104 and Part:BBa_J23105) are in the backbone BBa_J61002 (not pSB1C3) so we will get these synthesised as primers instead to reduce the amount of cloning we have to do. This will allow us to express promoter at different levels during design of experiment.

For low and high strength promoters:

BBa_J23104 and BBa_J23105 are in the plasmid BBa_J61002 and we want them in the pSB1C3

1. Cut at EcoRI and SpeI to cut out promoter from plasmid BBa_J61002.

2. PCR the promoter extracted.

3. Cut pSB1c3 at EcoRI and SpeI and ligate the promoter in.

4. Constructs ready to be used in Eut DoE experiments.

Eut Design of Experiment (DoE)

Basic aim: Optimise BMC formation

Modelling: Design of Experiments is used to find the optimal parameters using minimal tests by simultaneously changing multiple factors and making justified changes as the experiment is repeated. As the experiment is repeated, fundamental factors are identified and the next round of experiments hone in on these. By structuring our experiments and data in this way, we will be able to gain a deeper insight into to interactions and factors influencing our data. A general linear model can then be fitted (using JMP).

Response: Function of formation of microcompartments

Table 7: DoE Input Factors.
Name	Type of factor (categorical or continuous)	Difficulty (e, m, h, vh)	Range
Temperature	con		15 - 37 °C
pH	con		4.0 (E.coli strain M23) Optimum: 6-7 (O157:H7) Range: 4.4-9.0(O157:H7) E.coli BL21: 7.5-8.5 (Wang et.al, 2014)
Copy number of plasmid	cat
Tags x 5 + empty (6)	cat	e	n/a
Cell type	cat		BL21, DH5a
Strength of Anderson promoters	con		0.24, 0.51, 0.72
Time of BMC induction	con
Time of BMC harvesting	con
RPM in shaker (aeration)	con
Carbon source	cat
Iron conc	con
Phosphate conc	con
Magnesium conc	con
Carbon conc	con
Calcium conc	con
Organic solvent conc	con
Cell density	con
Secondary metabolite conc	con
ammonia/ammonium conc	con
Eut plasmid	cat		Maybe: plyss, pacyc duet, pET duet, pSB4A5
Concentration of inducers used for the promoters in the Eut operon	con		IPTG: 0-100 uM Tetracycline: 0-100 nM Arabinose: 0-20 mM (Lee et.al, 2011)

⍟ Actual Lab Work Record ⍟

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Team:Manchester/Notebook

Lab Book

🍀 Lab Plan 🍀

Polyphosphate Kinase (PPK) Fusion Protein

Introduction/background

Experimental Plan

Eut Microcompartment

Introduction/background

Experimental Plan

Localisation Tag Characterisation

Introduction/background

Selecting constitutive promoters from the registry

For low and high strength promoters:

Eut Design of Experiment (DoE)

⍟ Actual Lab Work Record ⍟

PPK

Eut

Tag