Difference between revisions of "Team:Hong Kong HKU/Results"

Revision as of 23:40, 18 September 2015

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.

PCR of gBlock (i)

We started PCR amplification after the arrival of both gBlocks and primers. Related figures are included in a table below. For the detailed preparation and concentration of reaction agents, please refer to our Notebook page.

	Stage 1	Stage 2			Stage 3
Time (mm:ss)	0:30	0:10	0:30	1:30	2:30	Infinite
Temperature (degree celcius)	98.0	98.0	Tx	72.0	72.0	4.0
Cycle	1	30			1	/

Table 2. The reaction figures for 1st PCR

For Tx, it is 3 degree Celsius higher than the lower Tm in that of the paired forward and reverse primers, to each gBlocks respectively.

- gBlock 1: 58.4 degree

- gBlock 2: 61.5 degree

- gBlock 3: 60.9 degree

- gBlock 4: 59.9 degree

- gBlock 5: 58.1 degree

The result product of our first PCR was run in 1% agarose gel and is provided below. gBlock 1, 3, 4 and 5 were fine with the band size though some non-specific band is also shown. Yet gBlock 4 was somehow vague to be seen under UV. gBlock 2 showed wrong band size. Gel purification for gBlock 1, 3, 4, 5 was done except for gBlock 5, and their DNA concentrations are low.

Fig. 3 The result of the 1st PCR.

The correct band size for gBlock 1-5 should be

- gBlock 1: 1120bp

- gBlock 2: 1889bp

- gBlock 3: 1500bp

- gBlock 4: 2000bp

- gBlock 5: 683bp

PCR of gBlock (ii)

PCR was performed once more for gBlock 1, 2, 3 and 4, but this time with DMSO added to eliminate any possible secondary structures that may account for gBlock amplification failure. PCR reaction settings were the same as PCR 1st. The result product of PCR 1st was run in 1% agarose gel and is provided below. gBlock 1, 3, 4 all showed correct band size and the DNA concentration increased compared to the 1st PCR after gel extraction. gBlock 2 still showed wrong band size.

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