Indigoidine Production - bpsA

Streptomyces verticillus ATCC15003 was cultivated and used for amplification of the svp PPTase. We tried to extract bpsA from S. lavendulae DSM40708 with a new set of primers (Primer RB11-20).

PCR results with various primers and PCR conditions suggested that the bpsA indigoidine synthetase gene is not present or mutated in S. lavendulae DSM40708. The amplification of svp was successful.
Photorhabdus luminescens laumondii TT01 has been shown to carry an indioidine synthetase called indC (Brachmann 2012). We will use indC instead of bpsA for the following project parts.

Indigoidine Production - indC

Since S. lavendulae DSM40708 does not carry the bpsA gene, we used indC from Photorhabdus luminescens laumondii TT01 instead (Brachmann 2012).
The PPTase Sfp, originating from the B. subtilis strain 168, has been shown to exhibit a broad substrate specificity (Nakano 1988) and thus was used to activate indC on pRB3. pRB3 is similar to previous plasmids (indigoidine synthetase and PPTase on pSB1C3) but contains a KpnI cutting site as a spacer between the first ribosome binding site (RBS) and the indC coding sequence, a BamHI cutting site as a spacer between the second RBS and the sfp coding sequence as well as a NheI cutting site at the end of sfp. Therefore, this plasmid can be used with both Gibson or CPEC and a classical restriction cloning assembly strategies.
If pRB3 is functional, we will continue testing various combinations of PPTases and indigoidine synthetases.

pRB3 is a functional pSB1C3 construct with the genotype [http://parts.igem.org/Part:BBa_R0010 lacI-Promoter]-[http://parts.igem.org/Part:BBa_B0034 RBS1]-KpnI-indC-[http://parts.igem.org/Part:BBa_B0029 RBS2]-BamHI-sfp-NheI-pSB1C3ΔmRFP1. The blue phenotype of pRB3-transformed E.coli TOP10 cells proofs production of the blue pigment indigoidine after 30 hours and incubation at 37 °C. Transformed cells expressing the indigoidine synthetase gene and producing indigoidine grow much slower than usual TOP10 cells. This effect was already reported by other groups (Owen 2011) and is supposed to be due to the slight toxicity of indigoidine.
Next week we will amplify other PPTases and assemble plasmids with combinations of indC, bpsA(pMM64), sfp, svp, svp(pMM65) and entD. The ladder is the endogenous PPTase of E. coli and considered to be inefficient in activating indigoidine synthetases (Takahashi 2007). We want to test and compare entD with the other PPTases when overexpressed on a plasmid.

Indigoidine Production – Testing PPTases

We wanted to test various PPTases on their functionality in activating the indigoidine synthetases indC and bpsA. The plasmids pRB3-10 represent all possible combinations of either indC or bpsA with the PPTases sfp, svp, svp (pMM65) or entD. We transformed E. coli TOP10 with all the plasmids.

All PPTases are capable of attaching the 4'-Phosphopanthetheinyl residue to the apo-form of the indigoidine synthetases as was observed by the blue phenotype of transformed E. coli TOP10 cells. The main focus was now on exchanging the T-Domain of the indigoidine synthetase indC to further proof the concept of modularity in the field of NRPS domains. We can easily determine which engineered versions of indC remain functionality by screening for blue colonies.

T-Domain Shuffling – Assembly of ccdB Plasmids

In the subsequent weeks we wanted to exchange the indC T-domain with several T-domains of other NRPS modules. These are modules derived from the Tyrocidine and Delftibactin pathway, from the E. coli NRPS entF and two NRPS modules of unknown function from P. luminescens itself.
pRB11 (derived from pKH1), pRB12 (derived from pKH2) and pRB13 (derived from pRB3) carry the ccdB gene with a RBS instead of their native T-Domain, thus they express an unfunctional indC and the ccdb toxin which kills E. coli TOP10 cells. We extracted the ccdB gene from pDONR (Invitrogen, [http://www.lifetechnologies.com/order/catalog/product/12536017 pDONR™221]). E. coli OneShot ccdb survival cells produce an antidote to ccdB and are able to survive upon transformation with the ccdB carrying plasmid. After exchange of the ccdB gene with a novel T-domain only positive E. coli TOP10 transformants survive and the background colonies will be diminished.

Assembly and/or transformations were inefficient, as blue colonies on each plate suggest that an excess of template was contained in the transformation mix.
A ccdB PCR screening of pRB12 was positive but transformation in DH5alpha, TOP10 and OneShot ccdb survival cells showed that the ccdB gene is unfunctional. In the following, all the insert from pDONR was used including a promoter and the ccdA gene.
Also we used a two plasmid strategy to test every possible combination of engineered indigoidine synthetases and the respective PPTases. We worked with indC on pSB1C3 and the PPTases on separate pSK3K3 derived plasmids.

Indigoidine Production – Testing PPTases

pRB3 contains indC and sfp and results in a blue phenotype after tranformation of E. coli MG1655, BAP1 and TOP10. pRB4-10 plasmids code for different combinations of either indigoidine synthetase indC or bpsA and the PPTase sfp, svp, svp (pMM65) or entD. Transformation of E. coli TOP10 showed that all combinations result in indigoidine production. This week we validated these plasmids using colony PCR screenings and Sanger sequencing (GATC-biotech, Konstanz).

The PCR screening was inconclusive but sequencing results showed that all combinations of indC or bpsA with sfp, svp, svp (pMM65) or entD have been assembled correctly.
These results are remarkable since entD was previously described as an inefficient activator of indigoidine synthetases (Takahashi 2007). In the following week we prepared separate PPTase plasmids for co-transformation with engineered versions of indC. Also, we included the delC PPTase from D. acidovorans SPH-1, which is the PPTase involved in delftibactin synthesis.

T-Domain Shuffling – PPTase Plasmids

This week we wanted to prepare plasmids for the T-Domain exchange experiments. We assembled four plasmids pRB15-18, each containing one PPTase on the pSB3K3-backbone with a kanamycine resistance. These plasmids should be used for co-transformations with the engineered indigoidine synthetase indC on a pSB1C3 plasmid pRB19 with a chloramphenicole resistance.

We successfully assembled pRB15-18.

BioBricks for the Registry

As a first set of BioBricks we chose the coding sequences of the four PPTases sfp, svp, entD and delC as well as the indigoidine synthetase indC. Since indC contains two internal RFC[10] cutting sites (EcoRI and SpeI), the DNA sequence had to be mutated for the part submission. Therefore we first assembled pRB21, which is identical to pRB3 but doesn't contain a PPTase.

We successfully assembled pRB21 and amplified the PPTases with the BioBrick RFC[10] prefix and suffix (Primer KH13-20). Next week we cloned the PPTases into the pSB1C3 backbone using restriction cloning and removed the EcoRI and SpeI cutting sites contained in indC using a CPEC mutagenesis strategy.

T-Domain Shuffling

We started inserting new T-Domains into indC to check their functionality. In total we first assembled 19 variants of pRB19, which are named pRB19-Txx. The appended three-letter code identifies the respective T-domain or TTE-domain and is listed on our plasmid page in the materials section.

Since PCR screenings of pRB19 were inconsistent, we started with replacing the T-Domain of pRB14, which contains sfp as a PPTase. Screenings and sequencing results showed that assembly and transformation were correct but all colonies except for that on the positive control retained a white phenotype. We tested all the other T-domains on the newly assembled pRB19 with every PPTase pRB15-18 next week.

T-Domain Shuffling – PPTase Plasmids

As the PPTase plasmids pRB15-18 showed red colonies after retransformation of a positively prepped colony, we picked new colonies and prepped them. We will do the co-transformation experiments with the new version of pRB15 but with the old ones of pRB16-18.

BioBricks for the Registry

This week we removed the two internal RFC[10]-cutting sites of indC (EcoRI and SpeI) on pRB21 using a CPEC approach to yield pRB22]. The modified indC (indC*) was amplified introducing the RFC[10] prefix and suffix (Primer KH21/22). The four PPTases and indC* coding sequences were cloned into pSB1C3 using standard BioBrick Assembly.

Due to faulty primer design of RB71, the resulting plasmid pRB22 was incorrect as well. We ordered a corrected version of RB78 and assembled the plasmids again in the following week.

T-Domain Shuffling

We again assembled pRB19 from pRB14 and did all T-domain-exchanges in this plasmid. After we picked and prepped positive colonies we performed co-transformations with pRB15-18 to see which PPTases are able to activate any of the T-domains inserted into indC.
To obtain experimental validation that the strategy to use two separate plasmids works and to verify that the PPTase plasmids are free of template backbone we co-transformed TOP10 cells with pRB21 and pRB15-18.
In parallel we assembled pKH4, which is derived from pRB14. The sfp coding sequence was replaced by a nonsense part of pMM65 using the cutting sites BamHI and NheI. In this way, we were independent from the success of the assembly of pRB19.

BioBricks for the Registry

After the corrected primer RB78 arrived we assembled pRB22 and cloned the BioBricks pKH6-10 for the part submission.

Domain Shuffling – PPTase Plasmids

The PPTase plasmids pRB15-18 have been reassembled with pSB3K3 from a different plate of the partsdistribution, using a CPEC approach. This week we validated those plasmids. Additionally, we assembled them by restriction cloning.

T-Domain Shuffling

Last week we assembled pKH4, which is a pSB1C3 derived plasmid coding for indC but lacking a PPTase. We validated the plasmid and used it as the template to assemble pKH5 in which the T-domain of indC were replaced by ccdB. In parallel and as a backup strategy, we assembled pRB19 using restriction cloning.
As a second backup strategy, we assembled pRB22 and pRB23 using CPEC with single fragments that were amplified from a P. luminescens cell pellet.

Both pKH5 and pRB22 were correct as seen by PCR screenings and sequencing. We used pRB22 to assemble pRB23, which is the ccdB-variant of pRB22, enabling to test every combination of T-domain in co-expression with the four PPTases.

T-Domain Shuffling

We prepared pRB23-T1 to -TTE19 and performed transformations in E. coli TOP10 as well as co-transformations with the four PPTase plasmids pRB15-18 and a pSB3K3-plasmid with a nonsense insert. This shows whether there is a dependency of T-Domain activation on the PPTases and if there are differences in indigoidine yield using different combinations of T-Domains and PPTases. The co-transformation with a pSB3K3 plasmid with a nonsense insert shows whether the smaller size of the colonies in our co-transformation experiments is due to the co-transformation itself or to the PPTases.

We sequenced every variant of pRB23-Txx and they were all correct. The cotransformation experiment showed, that some of the engineered indigoidine synthetases are still capable of producing the blue pigment. The indigoidine production strongly differs among the various T-Domains. All the combinations are shown in a big table in the detailled lab journal below.

T-Domain Borders

This week we tried to assemble pRB23-b11 to -b34, which are 12 pRB22-derived plasmids, in which the indC-T-Domain was exchanged by the bpsA T-domain. In every variant, the domain borders were defined differently. Those constructs were co-transformed with the four PPTase plasmids pRB15-18 as well as with a pSB3K3 plasmid containing a nonsense insert (Transformation strain: E. coli TOP10). The appearance of blue colonies on the plates showed which domain borders work best for the domain exchange between functionally related NRPS.

The cotransformations of the pRB23-bxx CPEC assembly products with the PPTase plasmids was not successful. Therefore, we prepared plasmid DNA of the pRB23-bxx variants and repeated the co-transformation experiments.

T-Domain Shuffling – PPTase Plasmids

As there is always leaky expression of indC* - due to the fact that E. coli TOP10 lacks the lac repressor and we use a lacPromoter - we assembled variants of the PPTase plasmids which also carry the lacI gene with a lacPromoter.

Engineered indC - Quantitative Assay

Since we want to characterize the indC*-variants and PPTases in a quantitative manner, we establish an assay for measuring the correlations between T-domains, PPTases, growth rate and indigoidine production using OD measurements. We take 96 well plates and measure absorption spectra of various indigoidine producing cultures.

BioBricks for the Registry

This week we prepared our parts for submission to the registry. We submitted the coding sequences of indC, sfp, svp, entD and delC, as well as expression constructs coding for indC with the exchanged T-domains that resulted in a blue phenotype. These are pRB22 and pRB23-T8, -T10, -T12, -T13, -T14. We also submitted our construct in which the indC-T-Domain is exchanged by the ccdB gene.
A table of all the parts we submitted can be found on the Parts site.

Engineered indC – Quantitative Assay

We started with the detailled characterization of our engineered indC-variants using a Tecan infinite M200 plate reader to measure the absorbance spectra of 52 liquid cultures of different (co-)transformants over time. The table in the detailled lab journal shows the loading scheme of our 96 well plate and the transformants we used.
Since a variant of indC, where the T-domain has been exchanged by the bpsA T-domain with the specific domain borders b31 resulted in a blue phenotype, we ordered primers to try other native T-Domains with these newly defined domain borders.

T-domain shuffling

Last week's co-transformation experiments showed that exchanging the indC-T-domain with the bpsA T-domain results in a functional indigoidine synthetase, provided specific domain borders were chosen. We now used this domain borders to also try the other native T-Domains T3-9 from B. parabrevis, D. acidovorans, P. luminescens and E. coli. We assembled the pRB23 variants T3 to T9 with the domain borders b31 and performed co-transformations with the PPtase plasmids pRB15-18.

We analyzed the data of the spectral absorption measurements versus time and compared the growth synamics and pigment production between different engineered indigoidine synthetases in interplay with different PPTases.

Controlling Indigoidine synthesis

Furthermore we co-transformed E. coli TOP10 with the lacI-Sfp-constructs and pRB22 to determine whether the indigoidine production is controllable.

The lacI-sfp-constructs were not functional because the transformed cells did not turn blue even if induced.
Instead we tried the E. coli NEB Turbo strain which overexpresses the lac repressor. In this way, leaky expression and thereby growth retardation might be prevented.

Domain Shuffling – Indigoidine Synthetase Conversions

Besides the delH4 T-domain only the plu2642 T-domain resulted in a functional indigoidine synthetase when introduced to indC. We used our NRPS-Designer to predict the domain sequence of plu2642 and the selectivity of its A-domain and found that it is a single-module NRPS with the domain sequence A, T, TE. Its A-domain exhibits glutamine specificity, analogous to our indigoidine synthetase. Also the basic domain sequence is similar, whereas the internal oxidation domain of indC is missing in plu2642.
Another NRPS module which shows glutamine specificity is tycC2 from the Tyrocidine pathway (further described in the <a href="https://2013.igem.org/Team:Heidelberg/Project/Tyrocidine">Peptide Synthesis Project Page</a>).
To take the domain shuffling experiment even one step further, we assembled every possible domain combination involving the indC oxidation domain and an A-, T- and TE-domain from indC. plu2642 or tycC2 and transform a given, naturally occuring NRPS module into a functional indigoidine synthetase.

Engineered indC – Quantitative Assay

This week we also repeated the spectral measurement of our engineered indC variants cotransformed with single PPTases to see which combination of PPTase and T-domain is most efficient in producing indigoidine.