Part:BBa_K4013020
ptac-RiboJ-puuc-kdc-aro8-B0015
This sequence contains an IPTG inducible promoter ptac-RiboJ，puuc，kdc，aro8，and a strong terminator B0015.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 3474
Illegal NgoMIV site found at 4281
Illegal NgoMIV site found at 5794
Illegal NgoMIV site found at 6148
Illegal AgeI site found at 1814
Illegal AgeI site found at 2088 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 2893
Functional Parameters
Metabolic pathway of IAA
In our experiments, two ways to convert L-tryptophan to Indole-3-acetic acid (IAA) are used (Figure 1). IPA and IAM pathway, respectively. In our experiment, IPA pathway and IAM pathway were used. The IPA pathway is what we finally need to use to produce IAA on cyanobacteria. It needs three different enzymes to turn L-tryptophan into IAA. On the other hand, IAM pathway needs two enzymes to make IAA. This pathway is used by Imperial College London in 2011 (BBa_K515100).
Enzymes of IPA pathway
IPA pathway requires three enzymes to convert L-tryptophan to IAA in three steps. tryptophan aminotransferase (taa1/aro8) transforms tryptophan into indole-3-pyruvic acid, then indolepyruvate decarboxylase (ipdc/kdc) transforms indole-3-pyruvic acid into indole-3-acetaldehyde, which is converted to indole-3-acetic acid through aldehyde dehydrogenase (aldh/puuc). Among them, aro8 and kdc genes come from yeast, while puuc comes from Escherichia coli.
Construction and function test of IPA Pathway circuits
The Ptac promoter, a functional hybrid promoter found in 2009, can be activated by IPTG. The expression can be increased by the increase of IPTG concentration. We referenced the Ptac promoter into the IPA pathway and these parts are transferred into pET28a vector (Figure 2).
In Figure 2, it shows that we had successfully assembled and made a IPA pathway that can be used in E.coli. We transfer the gene into E.coli DH5a ΔtnaA, a strain invented by Liks_China.
Salkowiski test is a test that can measure the concentration of IAA. It needs a reagent to test it. When we add the reagent into our IAA, we can see the color change. The redder the color, the more IAA it contains. We induced bacteria and did Salkowiski test every 24 hours. We did Salkowiski test for induction of 24 hours, 48 hours and 72 hours. Figure 3 shows our results. It can be seen that our IPA pathway has a high yield.
Transfer IPA Pathway to Cyanobacteria
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 in
By using the Golden Gate Assembly method, the parts are assembled, and we got two IPA pathways that can be used in cyanobacteria (Figure 6).Figure 6 shows that the pathways are constructed successfully. The two pathways are the pathway with p1321 promoter and the pathway with p0452 promoter (Figure 7). We also apply the Salkowiski test on these two pathways.
In Figure 7, 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.
IAA functional test
In order to prove the effect of our IAA, we designed an experiment. IAA is a plant growth hormone. We used the root growth of mung bean to reflect the effect of IAA. We also used MS media, a plant tissue culture medium containing 7g / L agar and 30g / L sucrose. The experiment was divided into three groups. The experimental group used MS medium containing IAA produced by IPA pathway to grow mung beans, while the two control groups were MS medium containing standard IAA ,and MS medium. Soak mung beans in water for 24 hours and wait for them to germinate. We adjusted the IAA concentration in MS medium to 1.5mg/l. Put the soaked mung bean on the culture medium, contact the root with the culture medium, and wait for the mung bean to grow in a light incubator for about a day. The following results are obtained (Figure 8).
In Figure 8, we can see that the mung bean grows naturally in the group with MS medium, and in other two groups which contains IAA, root growth was significantly inhibited. This is because a certain concentration of IAA will inhibit the growth of plant tissue. In the two groups with IAA, the growth of roots is similar, this means that the activity of our IAA is basically consistent with the standard IAA.
LC-MS analysis
To further prove that there is no difference between our IAA and standard IAA, we conducted LC-MS test. In Figure 9, the Zoom in shows the result of the two IAA. Both peak shows the molecular weight about 174. This is the IAA without a hydrogen atom. The molecular weight of IAA is 175.184. we believe that the two IAA is the same.
experiments for improvements
This year, we characterized an existed part BBa_K515100. We believe our part BBa_K4013020 is a functional improvement of the old one.
Enzyme and construction of IAM Pathway
There are two enzymes in the IAM pathway, iaaM and iaaH (amiE/ami1). L-tryptophan will be turned into indole-3-acetamide by iaaM, and then become IAA by iaaH. As shown in Figure 4, IAM pathway is used by two different promoters in our experiment. One of the promoters is pVeg2 and this formed a pathway (BBa_K515100) that Imperial College London had used in 2011. Another promoter is Ptac. The Ptac promoter is a functional hybrid promoter which is controlled by IPTG.
We did Salkowiski test on our Iam pathway and the original IAM pathway. From the results, the IAM pathway of pVeg2 promoter is indeed better than our PTAC pathway, but the gap is not large.
From the results of Salkowiski test (Figure 5), we can see the tilter of IAA produced by IPA pathway of Ptac (Ptac-IPA) is much higher than the IAM pathway of Ptac (Ptac-IAM). In the best group we induced, the titer of IAA in Ptac-IPA reached 154.27mg/L at 48 hour. By calculation, the yield is up to 88%. In contrast, in this group of Salkowiski test, although the IAA yield of Ptac-IPA was only 53% at 48 hours, the yield of Ptac-IAM was only 8%, and the yield of control group was 13%. The production of IAA in the IPA pathway is more than three times higher than the IAM pathway. This can prove the advantage of IPA pathway.
In these characterization experiments, we found that the efficiency of new IPA pathway converting Trp into IAA is about 4 times higher than IAA pathway.
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