Part:BBa_K1894001

shuttle plasmid which can replicate in cyanobacteria and E.coli

This part is originally a self-constructed shuttle plasmid that can both replicate in cyanobacteria and E.coli, but we extract its key sequence and link it to pSB1C3 plasmid backbone. The shuttle plasmid we constructed possesses the replication origins of E.coli plasmid and cyanobacterium plasmid, CaMV35S promoter, multiple cloning sites (MCS) and rbcS polyA terminator, Which makes it convenient to insert and express target gene and to screen out the recombinants. Ori site of cyanobacterium plasmid comes from indigenous plasmids pPbs extracted from Plectonema boryanum.

Usage and Biology

After we recover gene segment of GvpA1 from cloning plasmid pET30a(+), we then link GvpA1 gene with shuttle plasmid pPKE2. We introduce shuttle plasmid into our experiments because we need to transform both E.coli and microcystis aeruginosa during experiments. （The plasmid of microcystis aeruginosa itself does not contain selectable marker and cannot replicate in E.coli. Traditional plasmid vector used to transform E.coli cannot transform microcystis either. ）The shuttle plasmid pPKE2 we constructed and used possesses the replication origins of E.coli plasmid and cyanobacterium plasmid, CaMV35S promoter, multiple cloning sites (MCS) and rbcS polyA terminator subcloned from plasmid pKYLX一71.35, which makes it convenient to insert and express target gene and to screen out the recombinants. Ori site of cyanobacterium plasmid comes from indigenous plasmids pPbs extracted from Plectonema boryanum.

Characterization of the part BBa K1894001

The goal of the experiment is to prove that the complete shuttle plasmid we constructed can both replicate in E.coli and cyanobacteria. Therefore, five measures were taken, using methods of transformation, resistance screening and endonuclease identification.

• Use shuttle plasmid to transform E.coli.
• Positive clones were screened by kanamycin and plasmid DNA extracted is identified with restriction endonuclease.
• Conduct basic kanamycin resistance test of microcysis.
• Use shuttle plasmid to transform microcysis.
• Carry out kanamycin resistance screening after the shuttle plasmid was transformed into microcysis.

Considering that the shuttle plasmid contains kanamycin gene, we decided to carry out kanamycin resistance screening. Basic kanamycin resistance test of Microcystis aeruginosa shows that the algae cannot resist concentration of 5μg/mL and above on BG11. Therefore, we choose 10-15μg/mL kanamycin for screening. After shuttle plasmid is transformed into microcysis, transposon screening is carried out by 7-10 days of culturing on BG-11 medium which contains 15µg/mL of kanamycin.

First experiment: Validation of E.coli Transformation

After transformation of E.coli BL21(DE3), we successfully screened out the positive clones and extracted the recombinant plasmid (the shuttle plasmid) containing GvpA1 gene for restriction endonucleases identification.

Figure 1: Electropherogram of plasmid pPKE2 DNA

• A1:products after EcoRI digestion
  
• A2: products after BamHI digestion
  
• M: λDNA marker for Hind III digestion
  
• B1: DNA of recombinant plasmid after EcoRI and SphI digestion
  
• B2: Recombinant plasmid pPKE2
  
• M: λDNA marker for EcRI and HindIII digestion
  

The plasmid should have a length of 9.8kb after ligation with target gene GvpA1, which is verified and showed in A1. Gene segment of GvpA1 and pPKE2 both include BamHI sites and two separated segments with length 2.8kb and 7.0kb each will show up after BamHI digestion， which is verified in A2. When gene segment of GvpA1 is inserted into the plasmid, a new SphI site will show up between original EcRI-SphI segment (2.8kb). Therefore, two separated segments with length 1.3kb and 1,5kb each will show up after EcoRI and SphI digestion, which is verified in B1.

Second experiment: Validation of Microcystis transformation

Fig.2. growth condition of microcystis a week after transformation

Left: control group (no recombinant plasmid introduced)
Right: Microcystis of Recombination group appears on BG11 medium containing kanamycin

Methods

1)Transformation of E.coli cells

Preparation of chemically competent E.coli cells

• Inoculate 2ml LB broth with an aliquot (about 50μL)of the desired E.coli from the -80℃ freezer stock of cells.
• Incubate for 2h at 37℃.
• Add the 2ml seed cμLture to 250ml LB broth and grow at 37℃, shaking (about 200rpm) until OD600 of 0.3-0.4 (about 5 hours).
• Pre-cool the 50ml polypropylene tube, 80 EP tubes, CaCl2-glycerine (0.1mol/L CaCl2) and CaCl2- MgCl2 (80mmol/L MgCl2, 20mmol/L CaCl2). Set the centrifuge and prepare the ice tray.
• Transfer the bacteria into the 50ml polypropylene tube. Place it on ice for 10 minutes.
• Centrifuge at 4℃, 4100rpm for 10 minutes.
• Discard supernatant, then place the tube upside down to make sure trace liquid medium runs out.
• Add 30ml of pre-cooled CaCl2- MgCl2 per 50ml of initial liquid medium to resuspend bacteria cell pellet.
• Centrifuge at 4℃, 4100rpm for 10 minutes.
• Discard supernatant then place the tube upside down to make sure trace liquid medium runs out.
• Add 2ml of pre-cooled CaCl2 per 50ml of initial liquid medium to resuspend bacteria cell pellet.
• Transfer to EP tubes (50μL every tube) and store at -80℃.
  

2)Transformation of E.coli BL21(DE3)
Thaw competent cells rapidly by immersing frozen tubes in a 37℃ water bath after remove from 70℃ refrigerator. Draw about 50μL of the competent cells in a clean tube and add 5μL recombined plasmid, throw on ice for about 30 min. then put them in 42℃ for 90 second, immediately turn on ice for 2min. add 900μL LB medium and incubate in a roller drum at 37℃ fro 1hours. Took 100μL LB medium contain ampicillin, upside down when dried, put in 37℃ incubator over night. Validation by agarose electrophoresis after the plasmid DNA was extracted from transforming Escherichia coli.

3) Determination of the basic resistance of kanamycin

• Kanamycin was prepared in water at 0µg/mL、5µg/mL、10µg/mL、15µg/mL、20µg/mL and stored at -20℃ in 10-µL aliquots. An aliquot was thawed as needed and used once without refreezing.
• Equivalently draw the cyanobacterium at logarithmic growth stage and inoculate in BG-11 liquid medium containing kanamycin with all above concentration respectively, the cyanobacterium solution was cultured under the optimum conditions for one week and observation of growth status was carried out.
• It is determined that the cell of cyanobacterium cannot resist certain concentration of kanamycin if no algae community were found after one week of culture. Basic kanamycin resistance test of Microcystis aeruginosa shows that the algae cannot resist concentration of 5μg/mL and above on BG11. Therefore, we choose 10-15μg/mL kanamycin for screening after shuttle plasmid pPKE2 containing modified GvpA1 gene is transformed into microcysitis aeruginosa.

4) Transformation of microcysis

• Microcysis (FACHB-854) was cultured in BG-11 medium until OD730 reached 0.25-0.35 which is measured with a spectrophotometer.
• The cells were prepared for transformation by washing once in 10 mM NaCl and resuspending in BG-11 at 5 x 108 cells per ml (the cells were concentrated 10 times)
• Aliquots of cells (300 or 400 µL) were dispensed to glass test tubes or microcentrifuge tubes, and recombined plasmid DNA in TE buffer (10 mM Tris, 1 mM EDTA, pH 7.6) was added to a final concentration of 2 to 3 µg/ml. Plasmid DNA was stored as a 20 µg/ml solution, so that no more than 50µL was added to cells.
• The transformation mixtures were incubated for various times at 28 to 30°C in a constant-temperature chamber under standard conditions. The key parameter was the time of incubation of the cells with the donor DNA.

5) Microcysis transposon screening

• The cμLture mixture was coating on Millipore membrane (Φ9cm, pore diameter 0.45µm) covered on BG-11 medium and mircrocysis was cultured under standard condition with 20 hours.
• Transfer the membrane in BG-11 solid medium which containing 15µg/mL of kanamycin, This plasmid confers kanamycin resistance to transformed recipient cells. Single colony was found after 7-10 days of culture, select the growth colony and transfer to BG-11 liquid medium(containing 10-15µg/mL of kanamycin ), shake frequently and culture another 7 days.
• The cyanobacteria of best growth were selected and expansively cultured step by step (always containing 15µg/mL of kanamycin).

Contribution

Team: iGEM17_Nanjing_NFLS, 2017.10.20
Author: Qinyu Ge, Kaiwen Wu
Summary and Uploads:
We verified the part BBa_K1894001. Electrophoresis was carried out after cut with the restriction endonuclease, expected band was obtained. The obtained shuttle plasmid contain gene GvpA1 was cloned and transfected. Microcystis aeruginosa from TaiHu Lake were collected and treated, then cultured in laboratory as receptor cells, and transfected with the plasmid mentioned above. After cultured several days, results showed that many of the microcystis were sunk as expected, it can be seen in the figure below. This suggested that gene GvpA1 modified in the plasmid were expressed and the functions were preliminarily demonstrated. We plan to extracted the protein and verified with west blotting in further.

Figure: Modified gene in shuttle plasmid transfer to Microcystis aeruginosa collected from TaiHu Lake

Contribution

Group: HK_SSC, 2019
Author: LEE Hong Kiu, CHOI Justin Yuet Hei

Summary:

We would like to see how this shuttle vector affects the growth rate of E.coli BL21(DE3) and E.coli DH5α. We will plot a growth curve of transformants of this shuttle vector, and compare to that of transformants of Psb1c3, pET-blue-2 and PUC19.

Background:

It was found that plasmid sizes and different origin of replications have distinct effects on the growth of E.coli . Generally, larger plasmid sizes result in a slower growth rate in E.coli. While common vectors, including Psc1c3, pET-Blue2 and PUC19 have a size less than 4000bp, shuttle vector BBa_K1894001 has a vector size of 6913bp. It is unknown how the large plasmid size or the two ORI will affect the growth rate of E.coli. Data on how this shuttle plasmid affects the growth rate of E.coli has not been provided.

Purpose:

The purpose is to find out how this shuttle vector affects the growth rate of E.coli cells by comparing its growth rate to those of other common vectors. If the growth rate is found to be much slower than other vectors, it may not be suitable for cloning. This data will also give future user an approximate value of the cell yield they should expect.

Methods:

1. Preparation of Cells

E.coli competent cells were prepared using Inoue Method .

2. Calibration

We followed iGEM 2019 Plate Reader Abs600 (OD) Calibration protocol, s that we can estimate our number cells.

3. Cloning of shuttle vector shuttle vector BBa_K1894001

As shuttle vector BBa_K1894001 is of 6913bp, we could not synthesis it directly. We divided the plasmid into 3 fragments and assembled it using Gibson Assembly. The 3 fragments were designed to have 20-25bp overlap.

Figure 3: The construction of the BBa_K1894001 shuttle vector using Gibson Assembly

Our Sanger sequencing results have shown no undesired mutations in the junctions.

4. Transformation

10 ng of plasmid Psc1c3 and shuttle vector BBa_K1894001 were transformed into E.coli BL21(DE3) using iGEM’s transformation protocol. 10ng of plasmid Psc1c3, pET-Blue-2, PUC19 and shuttle vector BBa_K1894001 were transformed into E.coli DH5α using iGEM’s transformation protocol. Transformants were spread onto agar plates with respective antibiotics.

5. Inoculation

Single colonies from each plate were picked. They are inoculated in 3mL of LB with antibiotics for 16 hours at 37°C shaking at 250r.p.m.

6. Measurement

OD600 of the cell cultures were measured and diluted to OD600 ~ 0.1. Then, the diluted culture was inoculated at 37°C shaking at 250r.p.m. OD600 was taken exactly every 30 minutes interval. E.coli BL21 (DE3) with inserts of Psb1c3 (2070) and shuttle vector BBa_K1894001 (6913bp) are compared. E.coli DH5α with inserts Psb1c3 (2070 bp), pET-Blue-2 (3653 bp), PUC19 (2686 bp) and shuttle vector BBa_K1894001 (6913 bp) are compared. The experiments are performed in triplicates.

Assumptions made:

Assumption	Justification
1. The OD600 = 0.1 is equivalent to 9.3x106	This is according to the calibration curve performed using iGEM standard protocols: Calibration Protocol - Plate Reader Abs600 (OD) Calibration with Microsphere Particles V.2
2. The OD600 of the overnight culture is the maximum OD600.	OD600 remains constant staring from the stationary phase and 16 hours of incubation takes the E.coli to stationary phase .

Results

The below graph shows the results of the optical density measurements. The data shown is the average of three replicates.

E.coli DH5α

Time (h)	Psb1c3 (OD600)	PUC19 (OD600)	pET-Blue 2 (OD600)	BBa_K1894001 (OD600)
0	0.172	0.119	0.139	0.122
0.5	0.198	0.155	0.145	0.151
1.0	0.387	0.337	0.262	0.332
1.5	0.681	0.572	0.424	0.635
2.0	1.219	0.972	0.711	0.932
2.5	1.604	1.165	0.939	1.383
3.0	2.166	1.399	1.314	1.683
3.5	2.430	1.584	1.602	1.851
4.0	2.437	1.608	1.867	1.621
4.5	2.731	1.734	2.052	2.194
16.0	3.249	1.902	2.302	2.481

E.coli BL21 (DE3)

Time (h)	Psb1c3 (OD600)	BBa_K1894001 (OD600)
0	0.108	0.128
0.5	0.162	0.178
1	0.377	0.377
1.5	0.677	0.662
2	1.213	1.133
2.5	1.406	1.342
3.0	1.746	1.703
3.5	1.911	1.820
4.0	2.063	1.847
4.5	2.266	2.098
16.0	2.836	3.138

We further converted these data to the estimated number of cell counts using the iGEM standard protocols: Calibration Protocol - Plate Reader Abs600 (OD) Calibration with Microsphere Particles V.2. (Data not shown)

7. Curve Fitting

Gompertz model is the most frequently used sigmoid model to fit growth data in biology.

Figure 4: Gompertz Model

Curve fitting is done in Matlab.

Coefficients of the Gompertz function when f(x) represents OD₆₀₀ are as follows:

E.coli DH5α

	a	b	c
Psc1c3	3.218	4.380	0.7485
PUC19	1.970	3.660	0.7760
pET-Blue 2	2.537	4.801	0.6679
BBa_K1894001	2.476	4.218	0.7653

Figure 4: Graph of growth curve of E.coli DH5α transformants with plasmids Psb1c3, PUC19, pET-Blue 2, BBa_K1894001

https://www.desmos.com/calculator/tmrmerzvf3?embed

E.coli BL21 (DE3)

	a	b	c
Psb1c3	2.776	3.352	0.6195
BBa_K1894001	3.086	3.059	0.4932

Figure 5: Graph of growth curve of E.coli BL21 (DE3) transformants with plasmids Psb1c3, and BBa_K1894001

https://www.desmos.com/calculator/tmrmerzvf3?embed

Besides, we further compared the growth curve of E.coli DH5α and E.coli BL21(DE3) transformed with with BBa_K1894001. The graph is follow: T--HK_SSC--BL21NEB.jpg

We further converted this data into an estimated number of cell count. The data is as follow:

Figure 5: Comparison of BL21(DE3) and E.coli DH5α transformants

https://www.desmos.com/calculator/djfz6xqqew?embed
To our surprise, the final cell yield of E.coli BL21 (DE3) transformants was higher than that of E.coli DH5α.

E.coli DH5α

Time (h)	Psb1c3 (cells)	PUC19 (cells)	pET-Blue 2 (cells)	BBa_K1894001(cells)
0	1.60 x 107	1.11 x 107	1.29 x 107	1.14 x 107
0.5	1.84 x 107	1.44 x 107	1.35 x 107	1.41 x 107
1.0	3.61 x 107	3.14 x 107	2.44 x 107	3.09 x 107
1.5	6.35 x 107	5.33 x 107	3.95 x 107	5.92 x 107
2.0	11.4 x 107	9.06 x 107	6.63 x 107	8.69 x 107
2.5	15.0 x 107	10.9 x 107	8.75 x 107	12.9 x 107
3.0	20.2 x 107	13.0 x 107	12.3 x 107	15.7 x 107
3.5	22.7 x 107	14.8 x 107	14.9 x 107	17.3 x 107
4.0	22.7 x 107	15.0 x 107	17.4 x 107	1.51 x 107
4.5	25.5 x 107	16.2 x 107	19.1 x 107	20.5 x 107
15.0	30.3 x 107	17.7 x 107	21.4 x 107	23.1 x 107

E.coli BL21 (DE3)

Time (h)	Psb1c3 (cells)	BBa_K1894001(cells)
0.0	1.00 x 107	1.19 x 107
0.5	1.51 x 107	1.66 x 107
1.0	3.51 x 107	3.51 x 107
1.5	6.31 x 107	6.17 x 107
2.0	11.3 x 107	10.6 x 107
2.5	13.1 x 107	12.5 x 107
3.0	16.3 x 107	15.9 x 107
3.5	17.8 x 107	17.0 x 107
4.0	19.2 x 107	17.2 x 107
4.5	21.1 x 107	19.6 x 107
15.0	26.4 x 107	29.3 x 107

Conclusion:

In E.coli DH5α, shuttle vector BBa_K1894001 has a similar growth rate to pET-Blue 2 plasmid. The growth rate of BBa_K1894001is slower than that of Psb1c3 plasmid, but higher than that of PUC19. In E.coli BL21 (DE3), the growth rate of BBa_K1894001 shuttle vector was even faster than that of Psb1c3. In conclusion, the large plasmid size of BBa_K1894001did not adversely affect the E.coli growth rate as expected. Its growth rate is comparable to common vectors including Psb1c3, PUC19 and pET-Blue 2. The results show that BBa_K1894001 is still ideal for cloning.

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 5892
  Illegal NotI site found at 6620
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 6613
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1596
  Illegal NgoMIV site found at 1756
  Illegal NgoMIV site found at 4803
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 2676