Team:Hong Kong HKU/Results
RESULT
Design and Order gBlock
After the construction and optimization of our killing switch system that aims to work in E. coli BL21(DE3), we distribute them into 5 gBlock fragments, a synthetic double-stranded DNA product provided by IDT company. This strategy is chosen since our design included several new parts such as Cas9 and guiding RNA that target specific sequences. The whole construct also involved in numerous minor changes, thus traditional cloning and assembly of available biobricks cannot meet our needs.
Fig 1. Span of gBlock corresponding to the killing switch construct. For easy understanding, the length of DNA fragments in the figure are not in actual proportion.
These gBlocks are DNA fragments synthesized according to our order, and can be later assembled by Hi-Fi assembly. Fig. 1 shows the span of the gBlock fragments and their length in base pair. gBlock 1 initiates with prefix and gblock 5 ends with suffix, thus after assembly with pSB1C3 backbone, the whole construct forms a circular plasmid and can be transformed into targeted organisms.
Design and Order gBlock Primer
Fig. 2 Relative position of primers and gBlocks
While ordering the gBlock DNAs, we also designed and ordered the 10 forward and reverse primers that matched the 5 gBlocks respectively in order to amplify them through PCR. Fig. 2 illustrates the relative positions of gBlocks and primers in a circular plasmid. Since the forward/ reverse primers have overlapping regions on the preceding/ following gBlocks, after amplification all fragments would have overlapping sequences that enable Hi-Fi assembly. The sequences and working temperatures of each primers are provided in the table below. All primers were designed to have around 40 base pairs to provide enough specificity for the annealing DNA. Yet the arrangement exceeded the optimal length of primer design (which should be 18-24 bp) and the primers might have a higher chance to generate self-dimers or form secondary structure.
Overlaps | Oligo (Uppercase = gene-specific primer) 5'->3' | Anneals | F/R | 3' Tm | 3' Ta |
---|---|---|---|---|---|
gBlock 4 | tgggcctttctgcgtttataTACTAGAGTTTACGGCTAG | gBlock 5 | Fwd | 55.1°C | 58.1°C |
pSBCc3 | tgcagcggccgctactagtaAAAAAAAGCACCGACTCG | gBlock 5 | Rev | 58.3°C | 58.1°C |
pSB1C3 | attcgcggccgcttctagagTTATGACAACTTGACGGC | gBlock 1 | Fwd | 57.1°C | 58.4°C |
gBlock 2 | cgtgcaaataatcaatgtctCTAGTAATAGCAAAGTGTGAC | gBlock 1 | Rev | 55.4°C | 58.4°C |
gBlock 3 | aactgtcaaagttgttgatgAATTAGTAAAAGTGATGGGTCG | gBlock 4 | Fwd | 57.7°C | 59.9°C |
gBlock 5 | gctagccgtaaactctagtaTATAAACGCAGAAAGGCC | gBlock 4 | Rev | 56.7°C | 59.9°C |
gBlock 2 | caatctgatcgcattgtcgcTGGGTCTGACCCCTAACT | gBlock 3 | Fwd | 62.5°C | 60.9°C |
gBlock 4 | acccatcacttttactaattCATCAACAACTTTGACAGTTTG | gBlock 3 | Rev | 57.9°C | 60.9°C |
gBlock 1 | tcacactttgctattactagAGACATTGATTATTTGCACGG | gBlock 2 | Fwd | 58.5°C | 61.5°C |
gBlock 3 | aaagttaggggtcagacccaGCGACAATGCGATCAGATTG | gBlock 2 | Rev | 62.3°C | 61.5°C |
--- | CTCTAGAAGCGGCCGCGA | pSB1C3 | Rev | 68.1°C | 67.5°C |
--- | TACTAGTAGCGGCCGCTG | pSB1C3 | Fwd | 64.5°C | 67.5°C |
Table 1. Sequences of the 10 ordered forward and reverse primers. The lowercase letters correspond to the overlapping sequences and the capital letters represent the annealing sequences of the adjacent gBlock fragments. Tm stands for melting temperature, and Ta stands for annealing temperature.
4. If Taq polymerase is used instead of Q5, assemble all reaction components as followed on ice. Gently mix all the components before use. Collect all liquid to the bottom of the tube by a quick spin if necessary.
COMPONENT | 50 uL REACTION |
---|---|
Taq buffer | 5 uL |
dNTPs | 1 uL |
10 uM Forward Primer | 1 uL |
10 uM Reverse Primer | 1 uL |
Taq polymerase | 0.5 uL |
Template gBlock DNA | 2 uL |
PCR water | 39.5 uL |
5. Transfer PCR tubes to a PCR machine and begin thermocycling.
STEP | TEMPERATURE | TIME |
---|---|---|
Initial Denaturation | 98 degree Celsius | 30 second |
30 cycles | 98 degree Celsius | 10 seconds |
50-72 degree Celsius | 30 seconds | |
72 degree Celsius | 1.5 minutes | |
Final Extension | 72 degree Celsius | 2.5 minutes |
Hold | 4 degree Celsius |
Gel Extraction
- QIAquick Gel Extraction Kit
1. Cut the specific DNA fragments from the agarose gel with a scalpel.
2. Weigh the gel slice in an Eppendorf tube. Add 3 volumes buffer QG to 1 volume gel (100 mg gel to 100 uL).
3. Incubate the tube at 50 degree Celsius for 10 minutes. Vortex the tube every 2-3 minutes to help dissolve gel. The colour of the mixture is yellow.
4. Add 1 gel volume isopropanol to the sample and mix.
5. Place a QIAquick column in s provided 2-mL collection tube. Apply the sample to the QIAquick column and centrifuge for 1 minute. Discard the flow-through and place back the column back into the same tube (binding DNA).
6. Add 750 uL of buffer PE/EtOH to QIAquick column. Let the column stand for 4 minutes and centrifuge for 1 minute. Discard the flow-through and place back the QIAquick column back into the same tube (washing). Centrifuge the QIAquick column in the provided 2-mL collection tube for 1 minute to remove residual wash buffer.
7. Place the QIAquick column into a clean Eppendorf tube.
8. Add 30 uL of PCR water to the centre of the QIAquick membrane to elute DNA. Let the column stand for 1 minute and centrifuge for 1 minute.
HiFi DNA Assembly and Transformation
- NEBuilder HiFi DNA Assembly Master Mix
- Positive control provided: 2 overlapping dsDNA fragments for control assembly
length (bp) | Conc. (ng/uL) | pmol needed | Mass (ng) | Vol. (uL) | |
gBlock 1 | 1160 | 19.5 | 0.05 | 35.84 | 1.84 |
gBlock 2 | 1889 | 10 [stock] | 0.05 | 58.37 | 5.84 |
gBlock 3 | 1540 | 23.8 | 0.05 | 47.58 | 2.00 |
gBlock 4 | 2040 | 8.7 | 0.05 | 63.03 | 7.24 |
gBlock 5 | 723 | 36.5 | 0.05 | 22.34 | 0.61 |
pSB1C3 | 2070 | 25 [stock] | 0.05 | 63.96 | 2.56 |
1. Set up the following reaction on ice:
4-6 Fragment Assembly | Positive Control | |
---|---|---|
Recommended DNA Ratio | vector:insert = 1:1 | |
Total Amount of Fragments | 0.2-0.5 pmols X uL
|
10 uL |
NEBuilder HiFi DNA Assembly Master Mix | 20 uL | 10 uL |
Total Volume | 40.09 uL | 20 uL |