Part:BBa_K4013010
p2.4-101 Vector
This sequence is a PCC6803 cyanobacterial vector containing the SmR gene, which is resistant to spectaculin.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal SpeI site found at 4500
- 12INCOMPATIBLE WITH RFC[12]Illegal SpeI site found at 4500
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal SpeI site found at 4500
- 25INCOMPATIBLE WITH RFC[25]Illegal SpeI site found at 4500
Illegal NgoMIV site found at 2673 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1736
Illegal BsaI.rc site found at 1720
Functional Parameters
Synechocystis sp. PCC 6803 is a cyanobacteria which is widely used in synthetic biology. We used some native expression elements of PCC 6803 such as plasmids, promoters and terminators. pCB-SC101 (P2.4-101 vector)(BBa_K4013010) is a native plasmid backbone in PCC 6803 which contains spectinomycin resistance. We also used two different promoters for the IPA pathway, Psll1321 (BBa_K4013000) and Pssl0452 (BBa_K4013001). These two native promoters are relatively weak in PCC 6803. We made this choice because excessive IAA would inhibit plant growth. For the terminator, we used a strong terminator called TrrnB (BBa_K4013002).
Construction of expression system
By using the Golden Gate Assembly method, the parts are assembled, and we got two IPA pathways that can be used in cyanobacteria (Figure 2).
Figure 2 shows that the pathways are constructed successfully. The two pathways are the pathway with p1321 promoter and the pathway with p0452 promoter (Figure 3). We also apply the Salkowiski test on these two pathways.
In Figure 3, we use the data of induce 48 hours. The data shows that the IAA produced by IPA pathway with p0452 is higher than p1321, but not much. This is expected because promoter p0452 is stronger than p1321.
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