Molecular experiments notes of our project

Experiments overview spanning the project

June

6.21

1. Transformed plasmid pMG36e into E.coli Top10

6.22

1. Transformed plasmid pUC57-DRGN-1 into E.coli Top10
2. Grew pMG36e-transformed E.coli in LB broth

6.23

1. Isolation of pMG36e plasmid from bacteria solution
2. Grew DRGN-1 transformed E.coli in LB broth
3. Ran agarose gel for pMG36e. Result: no target band

6.24

1. Isolation of pUC57-DRGN-1 from bacteria solution
2. Double-enzyme digestion of isolated pUC57-DRGN-1 using EcoRI and PstI.
3. Ran agarose gel for confirmation. Result: confirmed.

6.25

1. Repeated the amplification experiment of pMG36e
2. Preparation of DH5ɑ competent cells

6.26

1. Selected single colony of pMG36e-transformed E. coli, and transferred in LB broth.
2. Amplified the E. coli DH5ɑ containing pNZ8148 plasmid

6.27

1. The extraction of pMG36e and pNZ8148 plasmid
2. Digestion of pNZ8148 using XbaI
3. Agarose gel electrophoresis of digested pNZ8148. Result: confirmed, and the extracted plasmid was in super-coiled form

6.30

1. Gel electrophoresis for pMG36e, pUC57-DRGN-1 and pUC19. Results：no band for pMG36e, the other two were successful.

---

July

7.18

1. Preparation of SGM17 broth, inoculation of L. lactis NZ9000 strain in medium.

7.19

1. Cultured another batch of L. lactis NZ9000 strain.

7.20

1. Preparation of L. lactis NZ9000 competent cells

7.21

1. Preparation of L. lactis NZ9000 competent cells.

7.22

1. Electroporation of pUC57-3*AMPs into L. lactis NZ9000 strain
2. Electroporation of acmA into L. lactis NZ9000 strain

7.23

1. Selected single colony of transformant and cultured in liquid medium for plasmid isolation.

7.24

1. Extraction of plasmid from transformant bacteria.
2. PCR the isolated plasmid using standard primers.
3. Agarose gel electrophoresis of PCR products. Result: no target band.

7.25

1. PCR the original pUC57-3*AMP, pUC57-acmA and isolated plasmid using standard primers (for Gibson Assembly)
2. Agarose gel electrophoresis. Result: PCR products from the original plasmid had target band; the product from isolated plasmid had no target band.

7.26

1. Digestion of pNZ8148 using PstI
2. Gel electrophoresis. Result: target band gained.

7.27

1. Gibson Assembly to integrate acmA in pNZ8148 and integrate 3*AMP in pNZ8148
2. Preparation of electroporation recovery solution
3. Electroporation and cultured the transformant on selection plate.

7.28

1. Checked the transformant plate. Result: Two plates (one for bacteria transformed with pNZ8148-3*AMPs and one with pNZ8148-acmA) had colonies but there were foreign bacteria; other plates either were much contaminated or didn’t have colonies at all.
2. Selected the single colony from plates and cultured them respectively in liquid media.

7.29

1. Checked the empty plate again. Result: still no bacteria grew
2. Streaked the bacteria from liquid culture on plates to check and separate the L. lactis from foreign contamination.

7.30

1. Checked the result of streaking contaminated liquid transformant culture (bacteria containing acmA and 3*AMPs).

---

August

8.01

1. Made another batch of Chl-resistant M17 plates.
2. Took L. lactis NZ9000 strain from liquid culture (preserved culture) and streaked on M17 Chl-resistant plates to check whether the original bacteria was contaminated. Result: not contaminated.
3. Measured the CFU of competent L. lactis—dilution of the competent cells and spread on M17 plates.

8.02

1. Electroporation of competent L. lactis with empty vector pNZ8148, and spread on M17 plates.

8.03

1. Selection of single colony of transformant that contains pNZ8148, and grew in liquid M17 broth.

8.04

1. Plasmid pNZ8148 isolation from transformant cell culture.
2. Transformation of recombinants (pNZ8148-3*AMPs and pNZ8148-acmA) into E. coli DH5ɑ.
3. PstI digestion of isolated pNZ8148 plasmid from L. lactis
4. Agarose gel electrophoresis. Result: pNZ8148 confirmed

8.05

1. Plasmid extraction of recombinants (pNZ8148-3*AMPs and pNZ8148-acmA) from E. coli DH5ɑ.
2. PCR the extracts of expt 1
3. Gel electrophoresis. Result: target band confirmed.

8.08

1. Transformation of sfGFP in E. coli DH5ɑ
2. Electroporation of L. lactis NZ9000 with pNZ8148
3. PCR the AIP sensing device generator (BBa_K2309003) from pUC57 vector.
4. Agarose gel electrophoresis. Result: no target band shown

8.09

1. Selected single colonies and inoculated into LB broth.
2. PCR 3*AMPs, acmA and AIP sensing device generator using standard primers.
3. Agarose gel electrophoresis. Result: no wanted band for each of the three products.
4. Inoculated the pNZ8148-transformed L. lactis in Chl-resistant M17 broth.

8.10

1. Extraction of pSB1C3-sfGFP from transformant E. coli
2. PCR sfGFP from the extracted plasmid.
3. Agarose gel electrophoresis. Result: confirmed.
4. Isolation of pNZ8148 from transformant L. lactis
5. Took Nanodrop measurement of DNA extracted. Result: no DNA

8.11

1. New standard primers arrived. PCR 3*AMPs, acmA and AIP sensing device.

8.13

1. Gel electrophoresis of PCR products. Results: target bands of three genes found.

8.15

1. Single enzyme digestion of pNZ8148 using PstI

8.16

1. Agarose gel electrophoresis of digested pNZ8148 plasmid
2. Gibson Assembly for linking 3*AMPs and acmA to pNZ8148 respectively.

8.28

1. Selected single colony from previous L. lactis that were transformed with pNZ8148. Inoculated into Chl-resistant M17 broth.

8.29

1. Isolation of plasmid from L. lactis
2. Using Nanodrop measured the concentration.
3. Gel electrophoresis

---

September

9.02

1. Preparation of solutions for L. lactis competent cells making.
2. Incubated L. lactis in M17 broth

9.03

1. Made L. lactis competent cells.

9.04

1. Diluted the competent cells and inoculated on M17 plates.
2. Counted the colony number and calculated the CFU concentration of prepared competent cells.

9.05

1. Electroporation of L. lactis competent cells with empty vector pNZ8148.

9.06

1. Calculated the transformation efficiency. Result: 1.11*104 CFU/ng.

9.09

1. Electroporation of L. lactis with pNZ8148-acmA and inoculated on Chl-resistant plates

9.10

1. Selected the single colony from plates, and inoculated in liquid media.

9.11

1. Diluted the bacteria solution and put in microplate reader. Measured the successive OD₆₀₀ for 1 day.

9.12

1. Induced the bacteria with nisin, and continued measuring the OD₆₀₀ value for 2 days.

9.14

1. Observed the growth curve of nisin-induced bacteria.

9.18

1. Made Chl-resistant M17 broth
2. Tested the chloramphenicol effectivity.

9.19

1. Digestion of pNZ8148 using PstI
2. Cleaned up the prior PCR products
3. Gibson Assembly

9.20

1. Transformed acmA in L. lactis by electroporation

9.21-9.24

Nisin-induced expression experiments (the same as the experiments done during 9.10-9.14).

9.25-10.10

Collaboration assignments

---

October

10.11

1. Amplification of linearized pSB1C3.
2. Gel electrophoresis
3. PCR products purification

10.12

1. PCR the LL-37-His-tag and GF-17 gene
2. Gel electrophoresis

10.13

1. PCR products purification
2. Digestion of linearized pSB1C3 using EcoRI and PstI

10.16

1. Double enzyme digestion of the LL-37-His-tag and GF-17 gene using EcoRI and PstI.
2. Ligated LL-37-His-tag with digested pSB1C3, and GF-17 with pSB1C3

10.17

1. Constructed Biobrick tandem repeats 3 xAMPs on plasmid pSB1C3;
2. Constructed Biobrick AcmA on plasmid pSB1C3;
3. Constructed Biobrick Quorum seneing (QS) on plasmid pSB1C3;
4. Agarose gel electrophoresis

10.18

1. Constructed Biobrick tandem repeats 3x AMP on plasmid pEt 28a (+);
2. Constructed Biobrick LL-37+ 6x His tag on plasmid pEt 28a (+);
3. Agarose gel electrophoresis

10.20

1. Induced LL-37+ 6x His tag protein expression with IPTG
2. Dot blot

---

Anti-microbial peptides assays notes of our project

June

6.20

1. Tested DRGN-1's effect on Escherichia coli (DH5α).

6.23

1. Tested DRGN-1's effect on Lactobacillus rhamnosus (LGG).

6.25

1. Tested DRGN-1's effect on Pseudomonas fluorescens.

6.28

1. Tested DRGN-1's effect on Escherichia coli DH5α, Staphylococcus aureus, and Pseudomonas fluorescens with overnight culture in 37℃ and 200rpm.

6.29

1. Prepared DRGN-1 solution gradients: 25ug/ml, 50ug/ml, 100ug/ml, 200ug/ml.
2. Prepared antibiotic (chloramphenicol) solution gradients: 25ug/ml, 50ug/ml.
3. Prepared Escherichia coli DH5α/ Staphylococcus aureus /Pseudomonas fluorescens solution gradients: x100, x1000.

6.30

Result:

1. DRGN-1 has effects on Staphylococcus aureus.
2. DRGN-1 has no effect on E. coli DH5α and Pseudomonas fluorescens strain.

---

July

7.01

Inhibition Ring of LL-37 on Staphylococcus aureus

1. Cultured Staphylococcus aureus in 37℃, 200rpm and for 8 hours.
2. Added LL-37 solution to discs.
3. Added Staphylococcus aureus to LB agar plates.

7.04

Inhibition Ring of Grammistin-Pp1 on Staphylococcus aureus

1. Cultured Staphylococcus aureus in 37℃, 200rpm and for 8 hours.
2. Added Grammistin-Pp1 solution to discs.
3. Added Staphylococcus aureus to LB agar plates.

7.07

Inhibition Ring of GF-17 on Staphylococcus aureus

1. Cultured Staphylococcus aureusin 37℃, 200rpm and for 8 hours.
2. Added GF-17 solution to discs.
3. Added Staphylococcus aureus to LB agar plates.

7.10

Inhibition Ring of the mixed AMPs on Staphylococcus aureus

1. Cultured Staphylococcus aureusin 37℃, 200rpm and for 8 hours.
2. Added the mixed AMPs solution to discs.
3. Added Staphylococcus aureus to LB agar plates.

7.13

Minimal Inhibitory Concentration (MIC) assay of LL-37 on Staphylococcus aureus

1. Cultured Staphylococcus aureusin 37℃, 200rpm and for 8 hours.
2. Pipetted same volume of Staphylococcus aureus and the diluted LL-37 solution and mixed.
3. Incubated overnight.

7.15

Minimal Inhibitory Concentration (MIC) assay of Grammistin-Pp1 on Staphylococcus aureus

1. Cultured Staphylococcus aureus in 37℃, 200rpm and for 8 hours.
2. Pipetted same volume of Staphylococcus aureus and the diluted Grammistin-Pp1 solution and mixed.
3. Incubated overnight.

7.17

Minimal Inhibitory Concentration (MIC) assay of GF-17 on Staphylococcus aureus

1. Cultured Staphylococcus aureus in 37℃, 200rpm and for 8 hours.
2. Pipetted same volume of Staphylococcus aureus and the the diluted Grammistin-Pp1 solution and mixed.
3. Incubated overnight.

7.19

Minimal Inhibitory Concentration (MIC) assay of the mixed AMPs on Staphylococcus aureus

1. Cultured Staphylococcus aureus in 37℃, 200rpm and for 8 hours.
2. Pipetted same volume of Staphylococcus aureus and the diluted AMPs mixture solution and mixed.
3. Incubated overnight.

7.22

1. Anti-biofilm assay of LL-37 on Staphylococcus aureus.

7.25

1. Anti-biofilm assay of Grammistin-Pp1 on Staphylococcus aureus.

7.29

1. Anti-biofilm assay of GF-17 on Staphylococcus aureus.

---

August

8.01

1. Anti-biofilm assay of the mixed AMPs on Staphylococcus aureus biofilms.

8.06

1. Growth curve measure of Staphylococcus aureus.

8.09

1. Growth curve measure of Staphylococcus aureus under the effect of the peptide LL-37.

8.12

1. Growth curve measure of Staphylococcus aureus under the effect of the peptide Grammistin-Pp1.

8.16

1. Growth curve measure of Staphylococcus aureus under the effect of the peptide GF-17.

8.19

1. Growth curve measure of Staphylococcus aureus under the effect of the mixed AMPs.

---

Interlab experiments

July

7.10

1. Prepared SOC medium and LB agar plates with chloramphenicol.

7.15

1. Calibrated the spectrophotometer and constructed the fluorescein calibration curve.

7.16

1. Tested competent cells.

7.17

1. Too many bacteria, made new competent cells.

7.19

1. Tested competent cells again.

7.20

1. Ordered new chloramphenicol.

---

August

8.1

1. Tested competent cells again and compared the newly arrived chloramphenicol with the old one.

8.2

1. Negative results.

8.3

1. Tested the effects of chloramphenicol with the concentration ranging from 1-50 μg/ml.

8.4

1. Negative results.
2. Tested the effects of chloramphenicol with the concentration ranging from 50-200 μg/ml.

8.5

1. Negative results.

8.7

1. Tested the borrowed chloramphenicol.

8.8

1. Positive results. Tested competent cells.

8.9

1. Positive results.
2. Transformation.

8.10

1. Inoculated bacteria to the liquid medium.

8.11

1. Diluted bacteria and measure the OD₆₀₀ by using the spectrophotometer, and measured fluorescence by using plate reader.