Wet Lab Notebook
Day 1 23/7/30
1. -Introduce the discipline in the laboratory, and review the general process of the experiment .
2.-Instructions of operating gauges - LB Medium ：
3.- Introduction of PCR(Polymerization Chain Reaction), each group starts to create the samples for PCR
4.-Introduction and operation of Agarose Gel Electrophoresis
5. Recovery of the products of PCR
Day 2 23/7/31
1.- Introduction about extraction of plasmids ：
2.-Introduction of the enzyme cutting, ligation, and transformation
3. Start the Enzyme Cutting Process. Each group creates one sample for vector PET28a and one sample for one of three target genes: UAO, allantoicase, and allantoinase.
*BamHI, XhoI, and NotI are restriction enzymes.
4.-Examine the product of enzyme cutting for vectors by electrophoresis.
Production and Laying of LB Agar Plates ：
6.-Extraction of Gel Electrophoresis Sample
7.-Start ligation process. According to the kind of target gene each group has made, all groups focus on constructing plasmids.
8.-Transformation of Bacteria
Day 3 23/8/1
1.- Start isolated colonies validation process.
2.-Introduction of constructing plasmid with the vector of pGex-4T-1.
3.-Start the linearization process for both vector(pGex-4T-1) and target genes(UAO,A-nase, and A-case). Each group creates 3 samples: two of them are samples of vector pGex-4T-1, and the rest is the sample of the target gene(UAO/ allantoinase/ allantoicase)
Reagents required ：
4.-Start to examine the product of PCR in isolated colonies validation process via agarose gel electrophoresis.
5.-Start shaking bacteria.
*Those bacteria will undergo transformation in the following procedures. When the products of PCR have undergone homologous recombination, the plasmids will be replicated in those bacteria.
Day 4 23/8/2
1.-Start to prepare for the examination of the product of PCR on 8.1 by electrophoresis. (Create the gel)
2.-Start to extract the products of PCR in 8.1.
*There are 3 samples for each group: two for the vector,pGex-4T-1, and one for the target gene. During the extraction process, one sample of vector and one sample of target gene are used.
3.-Examine the products received by electrophoresis.
4.-Start the homologous recombination process.
5.-Extract the plasmids from the bacteria cultured in 8.1.
6.-Start transformation.
7. Production and Laying of LB Agar Plates
Day 5 23/8/3
The tasks today are examining the products(plasmids) of the transformation and replication for the pGex-UAO plasmids and pGex-UAA plasmids, culturing and incubating the dH5alpha bacteria with target gene, extracting the target vectors from the incubated dH5alpha bacteria, transferring them to BL21 bacteria, incubating the bacteria, examining the vectors.
STEP I
-Prepare for and operate the isolated colonies validation ：
*Reagents for pGex-UAO
*Reagents for pGex-UAA
STEP II
-Incubate the BL21 bacteria.
STEP III
-After the examination for vectors pGex-UAO and pGex-UAA, start to retrieve the vectors from the dH5alpha cells by centrifuging.
STEP IV
-Transformation: transfer the vectors acquired to the incubated bacteria BL21.
STEP V
-Incubate the BL21 samples with vectors. (acquired from the transformation procedure)
STEP VI
-Use electrophoresis to examine the vector in BL21.
STEP VII
-Carry out IPTG Induction
Day 6 23/8/5
The purposes of today’s experiments are extracting the protein synthesized, purifying the protein synthesized to extract the enzymes(target products), and determining the optimum concentration for IPTG induction by measuring the concentration of IPTG used in each group and the concentration of protein in the products.
STEP I
-Exposes the proteins synthesized by ultrasonication.
STEP II
-Purify the proteins obtained by using Ni-NTA resin.
Day 7 23/8/6
The experiments today involve creating gels for SDS-PAGE and using SDS-PAGE to separate the proteins obtained in previous procedures, which demonstrates that the BL21 bacteria with the vector pGex-UAO and pGex-UAA is able to synthesize Uric acid oxidase, allantoinase, and allantoicase successfully.
STEP I
-Introduce the SDS-PAGE and create the gel for the electrophoresis.
STEP II
-Prepare for the sample used in SDS-PAGE analysis.
Step III
-Start the electrophoresis process.
-Construct the solid culture medium for Nissle 1917.
Step V
-Start the transformation process, transform the pGex-UAO vectors to the probiotics Nissle 1917.
Day 8 23/8/7
The main tasks today are examining the enzymatic activity of the enzymes produced by BL21 containing pGex-UAO and pGex-UAA, creating a standard curve that refers to the relationship between the concentration of uric acid and the effectiveness of the enzyme.
STEP I
-Prepare for the samples that are used to create the standard curve, which reflects the relationship between the concentration of the Uric acid and the absorbance of the product.
STEP II
-Prepare for the samples of testing UAO, construct the system of testing, and obtain the results.
Day 9 23/9/8
1. Amplification of pGEX-UAO, allantoinase, allantoicase:
2. Homologous recombination of pGEX-UAO, allantoinase, allantoicase:
Day 10 23/9/10
1. Validation of single clones and shaking culture:
2. Plasmid extraction and sequencing
Day 1 1 23/9/11
1. Transformation into BL21
2. Single clone validation and shaking culture
Day1 2 23/9/12
1. Scale-up cultivation
2. Protein induction
Day1 3 23/9/13
1. Protein extraction
2. Protein purification
3. SDS-PAGE