The handshake experiment tests how many functional bacteria can be transferred through handshakes: pipet some functional bacteria to 1st person’s hand, handshake with the 2nd person, and the 2nd person goes on to handshake with the 3rd person, until the 5th person, press their hands on the Petri dishes that contain kanamycin respectively in the end, cultivate these bacteria to the next day to see the results.
Notes: Academic pieces of literature prove E.coli safety:

Design of gene knockout

Principle of gene knockout

Construction of the gene knockout

1. PCR of Decaf pathway

Design of Decaf pathway

Construction of the Decaf Pathway
2. Electrophoretic PCR products
3. PCR products recycling
4. Product concentration test

1. The construction of plasmid of Decaffeination pathway and gene knockout through Golden gate and Gibson methods respectively as directed.
2. PCR of decaffeination pathway constructed plasmid through Golden Gate.
3. Electrophoretic PCR products
4. PCR products recycling
5. Product concentration test
6. Bacterial conversion of “Male” product(Decaffeination pathway), the bacteria were cultured to the next day for growth curve and phenotype verification, took one night for the colonies to appear.

7. PCR of gene knockout constructed plasmid through Gibson
8. Electrophoretic PCR products
9. PCR products recycling
10. Product concentration test
11. Bacterial conversion of “Female” product(Gene knockout), the bacteria were cultured to the next day for growth curve and phenotype verification, took one night for the colonies to appear.

1. Bacteria colonies were appeared

Three Petri dishes are on the way. The upper left Petri dish, the upper right Petri dish, and the middle and lower Petri dish are Decaf Pathway, Decaf Pathway, and up-KanR-down respectively.
2. Perform colony PCR

PCR result of KanR fragment

Decaf on the left and Up - KanR - Down on the right.

1. Plasmid extraction followed the instruction after colony PCR.

2. Enzyme digestion verification/ Enzyme-cut vertification, includes Electrophoretic/ run-glue verification. (KpnI and DpnI)
3. Electrophoresis of different endonuclease(Enzyme-cut vertification) The first two lanes have two bands, which are around 2500 and 2000-2500 base pairs. In the Snapgene simulation, the desired lengths are 2654 and 2103 bp respectively. The electrophoresis result corroborates the restriction enzyme digestion. Lanes 3 and 4 are under Dpnl digestion, which can be visualized in 2 bands around 1000 and 750 bp, as well as smaller bands around 250. This is a reasonable output, since various cutting sites of DpnI could be detected on the plasmid of pYB1s, the genome would be demerged to small pieces.

PccdK- up - Kan- down enzyme digestion verification.
Lane 2 and 3 are between 8000 and 12000 base pairs. In the Snapgene simulation, the desired lengths are 9155 bp. The electrophoresis result corroborates the restriction enzyme digestion.

pYB1s - ndmDBCAE enzyme digestion verification.
4. RED homologous recombination through electroporation
Goal: knocking out the gene of guaB in the desired plasmid
Method: Homologous recombination using RED system
RED system construction
Flanking: introduce a up-kan-down(5'-3') to substitude up-guaB-down fragment. penetrating the cell membrane: by using 1400 v 3 ms electroporation, a small amount of up-kan-down would have an opportunity to transpassing the phospholipid layer of the Bateria successively. construction of main components functional plasmid PKD46 will offer Exonuclease, Annealing Protein, and Inhibition of Host Exonucleases for the flaking fragment to bind on the plasmid and knock out the guaB gene with an antibiotic.

Electroporator
Fig 1: Electroporator
Finally, 100 ul of electropored bacteria was spread evenly on the agar plate and 50 ul of xanthine was added to test the conditional growth and the success of target fragment incorporation.

1. Phenotypic verification preparation(gel of anything with xanthine and caffeine)
Bacterial Petri dishes made of different materials, from left to right are oolong tea, jasmine oolong tea, green tea, coffee, creatine, and C4 - Preworkout.


Growth curve on the Microplate reader.
2. Transfer three types of strains into three types of nutrient medium at aseptic bench(LB,LB+Xan,LB+caf)
3. 37 degrees celsius shake cultivation
4. Growth curve tested by Microplate reader.

1. PCR amplification of the plasmid of the Temperature-Controlled Cell-Free Expression System, including vector, restriction enzyme DpnI, and lambda repressor (Control/regulation)
2. Electrophoretic PCR products
3. PCR products recycling

PCR samples
4. Product concentration test

1. The construction of plasmid of pY38a-DpnI(Temperature-Controlled Cell-Free Expression System) through Gibson method as directed.
2. PCR the plasmid constructs
3. Electrophoretic PCR products

4. PCR products recycling
5. Product concentration test


6. Bacterial conversion of py38a-DpnI(Temperature-Controlled Cell-Free Expression System) product, the bacteria were cultured to the next day for growth curve and phenotype verification, took one night for the colonies to appear.

1.No colony grows at the first connection

2. It is connected again and then chemically transformed
3. Growth curve and phenotypic prediction

4.Colony PCR did not produce the correct bands

5.It is connected again and then chemically transformed

7.Perform colony PCR

Graph of electrophoresis for colony PCR.
8.Growth curve and phenotypic prediction

Schematic diagram of temperature sensitivity experiment of E. coli.

1. Plasmid extraction

Check plasmid concentration.
2. Enzyme digestion verification

3. Phenotype verification