Team:DTU-Denmark/SPL

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Synthetic Promoter Library


Figure 1: An SPL designed on the basis of randomizing both the spacer sequences surrounding the consensus regions (-35 and -10 regions) as well as randomizing two bases within each of the consensus regions is illustrated. N stands for 25% each of A, C, G and T, while S stands for 50% each of C and G, and W stands for 50% A and T.

Figure 2: The linear BioBrick plasmid backbone with SPL inserted between the EcoRI and XbaI sites of the BioBrick prefix is illustrated.

Figure 3: The primer binding sites on a BioBrick plasmid backbone as well as the final linear plasmid backbone that is generated by the PCR is illustrated.

Table 1: illustrates the Tm of the SPL primers. IDT DNA oligo analyzer was used in order to calculate the Tm.
PrimerTm - °C
I) Primer SPL Suffix-F62.1
II) Primer SPL Prefix-R-01 59.8
III) Primer SPL Prefix-R-0260

Table 2
PCR substratesVolumes - μL
Total volume50
Phusion Polymerase (0,02 U/μL)0.5
x5 Phusion HF buffer10
dNTP's (5μM)2
Primer SPL Suffix-F (10μM)1.25
Template - BioBrick plasmid backbone1
ddH2O33.5

Table 3
Cycle stepTemperature - ºCTimeCycles
Initial denaturation9830 sec1
Denaturation9810 sec-
Annealing63*30 sec20-25
Extension7230 sec / kb-
Final extension7210 min1
Hold4forever1

Table 4: Prefix SPL primers that SHOULD be used depending on which BioBrick plamid backbone is selected for amplification is illustrated.
BioBrick Plasmid BackbonePrimer IIPrimer IIISizes - bps
pSB1A3+-2157
pSB1AC3+-3055
pSB1AK3+-3189
pSB1AT3+-3446
pSB1C3+-2072
pSB1K3+-2206
pSB1T3+-2463
pSB2K3+-4425
pSB3C5-+2738
pSB3K5-+2936
pSB3T5-+3252
pSB4A5-+3395
pSB4C5-+3221
pSB4K5-+3419
pSB4T5-+3735

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