Basic Molecular Biology Techniques:

PCR using Q5 Hi-Fi DNA polymerase Master Mix

Primer 1: 5’-GCACTCACCATGGGTACTGCAGAATTCGCGGCCGCTTCT-3’

Primer 2: 5’-TAGTGGTACCGCATGCCTGCACTGCAGCGGCCGCTACT-3’

Reaction setup:

The template DNA should keep an amount of 1-1000 ng

Thermocycler conditions

Clarification:

1. The sequences in red are where the primers anneal with the biobrick affixes. And the 5’overhangs side with the sequences in red are designed for Gibson Assembly with linearized pNZ8148 digested by PstI due to a 21-nucleotide overlap between them.
2. The reason of setting two different cycles is that, at the beginning of PCR, the annealed parts between primers and templates are only sequences in red. As PCR proceeds, 5’overhangs are added to the entire genes, so the rest reaction of PCR becomes the one between the whole primers and the whole genes, because of which the Tm varies compared to the former.

Applications in this project: Amplification of BBa_K2309028, BBa_K2309003 and BBa_K2309004

PCR purification using QIAquick PCR purification kit

1. Add 500 μl Qiagen buffer PB
2. Spin through a column twice, discard flow-through
3. Wash 1x with 700 μl buffer PB
4. Wash 2x with 760 μl buffer PE
5. Discard liquid, spin dry at 17000g for 3 min
6. Elute into a new tube twice with 50 μl of TE (100 μl total)

(Can be also referred to from iGEM protocol)

Gel extraction

Gibson Assembly

Digestion (NEB enzymes)

Ligation

Transformation of plasmids in E.coli DH5ɑ

Isolation of plasmid in E.coli DH5ɑ

Preparation of competent Lactococcus lactis

Electroporation for L. lactis

Plasmid DNA isolation from Lactococcus lactis

Anti-microbial peptides assay:

Nisin induction of gene expression in Lactococcus lactis:

1. Add 100μl of LUDOX into wells A1, B1, C1, D1.
2. Add 100μl of H₂O into wells A2, B2, C2 D2.
3. Measure absorbance 600 nm of all samples in a microplate reader and record the data.
4. Add 1ml of LUDOX into cuvette for the measurement of OD₆₀₀ in spectrophotometer.

Fluorescein fluorescence standard curve

1. Spin down fluorescein stock tube to make sure pellet is at the bottom of tube.
2. Prepare 2x fluorescein stock solution (100μM) by re-suspending fluorescein in 1ml pf 1x PBS.
3. Dilute the 2x fluorescein stock solution with 1x PBS to make a 1x fluorescein solution and resulting concentration of fluorescein stock solution 50μM.
4. Add 200μl of 1x fluorescein stock solution into well A1, B1, C1 D1 of 96-well plate and 100μl of PBS to A2, B2, C2, D2…A12, B12, C12, D12.
5. Transfer 100μl of solution from A1 into A2 and mix by pipetting up and down three times, and then continue the same procedure and transfer from A2 to A3, from A3 to A4……Finally, transfer 100μl from A11 into liquid waste. Repeat dilution series for rows B, C, D.
6. Measure fluorescence of all samples in a microplate reader and record the data.

Cell measurement

1. Day 1: Transform Escherichia coli DH5α with these following plasmids:
   • Positive control
   • Negative control
   • Test Device 1: J23101+I13504
   • Test Device 2: J23106+I13504
   • Test Device 3: J23117+I13504
   • Test Device 4: J23101.BCD2.E0040.B0015
   • Test Device 5: J23106.BCD2.E0040.B0015
   • Test Device 6: J23117.BCD2.E0040.B0015
2. Day 2: Pick 2 colonies from each of plate and inoculate it on 5ml LB with 25μg/ml chloramphenicol for about 17 hours (3:00pm to 10am, next day), at 37℃ and 220rpm.
3. Day 3: Cell growth, sampling, and assay
   1. Take 200μl of each overnight culture mixed with 800μl water to make a 5-fold dilution.
   2. Measure OD₆₀₀ of the overnight cultures and record the data.
   3. Dilute the cultures to a target OD₆₀₀ of 0.02 in 12ml LB medium + Chloramphenicol in 50ml falcon tube.
   4. Incubate the cultures at 37°C and 220rpm.
   5. Take 500μl samples of the cultures at 0, 2, 4, and 6 hours of incubation.
   6. Place samples on ice.
   7. Take 100μl of each sample into 96-well plate to get values of fluorescence and OD₆₀₀. For each sample, there were 4 replicates.

We laid out sample as shown in following picture:

---

Result

The OD₆₀₀ reference

Fluorescein fluorescence standard curve

Fig 1.    The fluorescence standard curve of fluorescein
The measured fluorescence was plotted against the concentration of fluorescein.

The fluorescence signal increased 150 A.U. with the concentration of fluorescein increased 1μM at low concentration, from 0 to 20μM, and the slope of the curve was gradually decreasing as the concentration increased.

Cell measurement

Fig 2.    OD₆₀₀ against the incubation time
This figure showed the OD₆₀₀ of 16 samples with different devices during the incubation.

The growth rate of DH5α transformed with device 1 was significantly less than that of DH5α transformed with other devices, while this might have been an effect of the gene expression of device 1, but this can also happen due to human error.

Fig 3.     Fluorescence signals from different devices over time
In this figure, we give a curve of fluorescence signal of each sample at specific points (0, 2, 4 and 6 hours).

The culture of DH5α transformed with device 2 produced the highest fluorescent signal and followed by device 4 less than the positive control.

Fig 4.     GFP productivity of different devices

The blue, orange, grey and yellow bars correspondingly represented the ratio of GFP concerntration to OD₆₀₀ value at 0, 2, 4, 6 hours. The concertation of GFP was converted from fluorescence signal (in Figure 3), with calibration curve in Figure 2.

---

Discussion

We observed that, among the 6 combinations of these 3 promoters (J23101, J23106 and J23117) and 2 RBS (B0034 and BCD2) in various devices, devices 2 and 4 produced high fluorescence signals in the bacterial cell population and device 1 had the strongest ability to produce GFP per cell which was companied with low growth. Based on the plot of [GFP]/OD₆₀₀, either with RBSs B0034 or BCD2, the rank of the promoter efficiency (from strong to weak) is J23101, J23106 and J23117. When the promoters are the same, the strength of BCD2 is weaker than that of B0034. It was quite typical that, although the population of bacterial cells with device 1 had relatively low GFP level, the [GFP]/OD₆₀₀ of device 1 was the highest (except 0h) among all the samples. However, when merely considering the total quantity of GFP in bacterial population, obviously device 1 is not a good choice to produce GFP. That may be due to the overexpression of GFP in comsuming too many resources, leading to the lack of materials for cell growth.