Team:XJTLU-CHINA/Notebook

Lab book

Lab Book

Molecular experiments notes of our project

Experiments overview spanning the project

June

6.21

  1. Transformation of pMG36e into E.coli Top10

6.22

  1. Transformation of pUC57-DRGN-1 into E.coli Top10
  2. Grow pMG36e-transformed E.coli in LB broth

6.23

  1. Isolation of pMG36e plasmid from bacteria solution
  2. Grow DRGN-1 transformed E.coli in LB broth
  3. Run 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. Run agarose gel for confirmation. Result: confirmed.

6.25

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

6.26

  1. Select single colony of pMG36e-transformed E. coli, and transfer in LB broth.
  2. Amplify 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 is in super-coiled form

6.30

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

July

August

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. Making Chl-resistant M17 broth
  2. Testing the chloramphenicol effectivity.

9.19

  1. Digestion of pNZ8148 using PstI
  2. Clean 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. Construcedt 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

Interlab experiments

July

7.10
Made SOC medium and LB agar plates with chloramphenicol.
7.15
Calibrated spectrophotometer, constructed fluorescein calibration curve.
7.16
Tested competent cells.
7.17
Too many bacteria, made new competent cells.
7.19
Tested competent cells again.
7.20
Ordered new chloramphenicol due to the still large number of the bacteria on plates

August

8.1
New chloramphenicol arrived. Tested competent cells again and compared new chloramphenicol with old one.
8.2
Negative results.
8.3
Tested chloramphenicol concentration from 1-50 μg/ml.
8.4
Negative results.
Tested chloramphenicol concentration from 50-200 μg/ml.
8.5
Negative results.
8.7
Tested borrowed chloramphenicol.
8.8
Positive results. Tested competent cells.
8.9
Positive results. Started Transformation.
8.10
Inoculated bacteria to liquid medium.
8.11
Diluted bacteria and measured OD600 using spectrophotometer and measured fluorescence using plate reader.

Collaborators and Supporters

Location

Rm 363, Science Building
Xi'an Jiaotong-Liverpool University
111 Ren'ai Road, Suzhou, China
215123

Get in touch

emali

igem@xjtlu.edu.cn

XJTLU-CHINA iGEM 2017