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Indigoidine. Proving Modularity of NRPS by Shuffling Domains.

Week 5

Indigoidine Production - bpsA

At the beginning of our wetlab phase, we wanted to transform E. coli cells with plasmids containing an indigoidine synthetase and a 4'-Phosphopanthetheinyl-transferase (PPTase) to see whether we can observe blue colonies on our plates as this has been reported by groups working with indigoidine synthetases (Takahashi 2007, Brachmann2012).
We focused on the work of Marius Müller et. al. in 2012 (Muller 2012). The group used the bpsA indigoidine synthetase (blue pigment synthetase A) from S. lavendulae ATCC11924 (Takahashi 2007) and the PPTase svp from S. verticillus ATCC15003 (Sanchez 2001) to establish both a fluorescence and a chromophore based reporter assay for mammalian cells by expression of bpsA and svp which results in production of a blue pigment/ fluorophore. The group kindly supported us by sending two of their constructs, namely the pET derived expression vectors pMM64 carrying the bpsA indigoidine synthetase with an ampicillin resistance gene and pMM65 carrying svp and a kanamycin resistance gene. Both bpsA and svp rom the Fussenegger lab are codon-optimized versions for expression in eukaryotic cell lines.
We transformed competent E. coli TOP10 with each plasmid to prepare plasmid DNA and perform a cotransformation of an E. coli Rosetta strain with both plasmids. Transformed cells which carry both plasmids should produce the blue pigment indigoidine.

Unfortunately, there were no blue colonies after the co-transformation. We repeated the experiment under various growth conditions; i.e. we used different incubation temperatures, light conditions, shaking and addition of ascorbic acid, which was reported to stabilize indigoidine in liquid cultures (Muller 2012).

Week 6

Indigoidine Production - bpsA

This week we repeated the co-transformation of both the E. coli Rosetta and the BAP1 strain with the bpsA (pMM64) and the svp (pMM65) plasmids. The E. coli BAP1 strain is a variant of E. coli BL21(DE3) and contains the sfp PPTase from B. subtilis 168 (Nakano 1992, Quadri 1998) and a T7-promoter, brought in using a genomic integration approach (Pfeifer 2001). The Pfeifer lab kindly supported us by sending the BAP1 strain.
After induction, liquid cultures of positive BAP1 transformants should turn blue.

Both strains still didn't turn blue when grown and induced on plate and in liquid culture, even after some days with different growth conditions. We continued with the validation of the pMM-plasmids.

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Week 7

Indigoidine Production - bpsA

Since we couldn't observe indigoidine production using pMM64 and pMM65 in the previous two weeks we validated the pMM-plasmids using restriction digests.

The restriction digest of pMM65 was correct but pMM64 didn't show the expected bands on an agarose gel. We continued trying to amplify the bpsA and svp gene in a PCR screening.

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Week 8

Indigoidine Production - bpsA

We performed PCR screenings of the pMM64 (bpsA) and pMM65 (svp) plasmid for validation.

The PCR screenings showed that the pMM-plasmids are correct. We designed primers to assemble our first [http://parts.igem.org/Part:pSB1C3 pSB1C3] derived plasmid pKH1, which carries both the bpsA and the svp gene with the lacI-Promoter [http://parts.igem.org/Part:BBa_R0010?title=Part:BBa_R0010 BBa_R0010] and the ribosome binding site [http://parts.igem.org/Part:BBa_B0034?title=Part:BBa_B0034 BBa_B0034] before both the bpsA and the svp gene, respectively..

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Week 9

Indigioidine Production – bpsA

This week we assembled our first [http://parts.igem.org/Part:pSB1C3 pSB1C3] derived plasmid pKH1, which carries both the bpsA and the svp gene with the lacI-Promoter [http://parts.igem.org/Part:BBa_R0010?title=Part:BBa_R0010 BBa_R0010] using a Gibson (Gibson 2009) and a CPEC (Quan 2009) cloning strategy. We replaced the [http://parts.igem.org/Part:BBa_E1010 mRFP1 coding sequence] with the bpsA and svp coding sequences with the [http://parts.igem.org/Part:BBa_B0034 RBS B0034], respectively. Thus we assemble three fragments in total, which are pSB1C3ΔmRFP1 (Primer NI9/10), bpsA (Primer NI1/6) and svp (NI7/8).
With this plasmid it is possible to produce indigoidine in E. coli TOP10 without IPTG induction, since TOP10 cells lack the lac repressor gene.

As the PCR amplifications of all fragments for the assembly of pKH1 were positive, we could perform both the Gibson and CPEC assembly for pKH1.
After IPTG induction, co-transformation of BAP1 with the originalpMM64 and pMM65 yielded blue liquid cultures. We compared the blue color using four samples induced by different amounts of IPTG. We couldn't detect significant correlation between indigoidine production and varying IPTG concentration.
Since we have had problems with the plasmids received from Zurich and we wanted to use the original gene rather than a codon optimized version, we planned to extract the bpsA indigoidine synthetase gene directly from the organism it originates from, namely Streptomyces lavendulae subsp. lavendulae strain ATCC11924 (Takahashi 2007). As the DSMZ (German Collection of Microorganisms and Cell Cultures) doesn't provide this specific strain any longer, we used another S. lavendulae subsp. lavendulae strain ([http://www.dsmz.de/catalogues/details/culture/DSM-40708.html?tx_dsmzresources_pi5%5BreturnPid%5D=304 DSM40708]) expecting the bpsA gene to be present as well. We cultivated S. lavendulae in both YEME medium (Kieser 2000) and GYM medium ([http://www.dsmz.de/microorganisms/medium/pdf/DSMZ_Medium65.pdf DSMZ medium No 65]) at 28 °C.

Week 10

Indigoidine Production - bpsA

For the PCR amplification of native bpsA, Streptomyces lavendulae subsp. lavendulae DSM40708 was grown on GYM agar plates. We wanted to use the native indigoidine synthetase bpsA and the PPTase svp from pMM65 and to assemble pRB1, which is similar to pKH1. In this new assembly approach, we amplify the bpsA gene from the S. lavendulae genome instead of using a codon optimized sequence. Furthermore the svp coding sequence was placed behind a weaker promoter ([http://parts.igem.org/Part:BBa_B0029 BBa_B0029]) since the PPTase has to activate every indigoidine synthetase only once and thus is not required in huge amounts (Lambalot 1996).
In the subsequent weeks we wanted to exchange the Thiolation-domain (T-domain) of bpsA with T-Domains of other NRPS modules and show that it can be activated by various PPTases. We wanted to find out whether there are differences in the PPTases' efficiency concerning the activation of engineered indigoidine synthetases.

The PCR amplification of bpsA was unsuccessful. We will try again with a new set of primers.
In addition we will try to amplify the native svp PPTase from Streptomyces verticillus ATCC15003, which has been described in previous studies (Sanchez 2001).

Week 11

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.

Week 12

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.

Week 13

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.

Week 14

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.

Week 15

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.

Week 16

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.

Week 17

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.

Week 18

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.

Week 19

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.

Week 20

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.

Week 21

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.

Week 22

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.

Week 23

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.

Lab book

We got two plasmids from the Fussenegger group at the ETH Zurich containing a codon optimized version of bpsA

(pMM64) and svp (pMM65), respectively, for expression in HEK 297-3 cells [Muller 2012]. The first goal is to

transform the plasmids into competent E. coli (Rosetta) and check functionality of the plasmids in our cells.

Indigoidine production with pMM-plasmids I (Ilia)

After Transformation cells are grown first on Amp medium and thereafter on Kan medium.

transform competent Rosetta with 225 ng pMM065 and 253.5 ng pMM064
plate on Amp plate
pick colonies from Amp plate, make liquid cultures in LB + Kan + IPTG(1 mM)
Evening: no growth in liquid culture => prepare ON culture in LB+Amp
Put ON culture in incubator
prepare competent Rosetta-pMM064 from ON culture
transform with 225 ng pMM065
plate on Kan+IPTG plate
no growth on Kan plate

Results and Discussion

There were no blue colonies. Transformation will be repeated and medium will be provided with both antibiotics.

Lab book

Transformed Rosetta with pMM64 and pMM65 didn't show a blue phenotype. Transformation will be repeated.

Indigoidine production with pMM-plasmids II (Ilia)

one blue colony on plate
make ON culture in LB + Amp + Kan + IPTG(1 mM)
transform BAP1 with 169 ng pMM064, plate on LB+Amp+IPTG
ON culture opaque, but colorless
plate contains BAP1-pMM064 colonies, but colorless
prepare fresh IPTG
inoculate 5 ml LB + Amp + Kan + IPTG(10 mM) with 1 ml from Rosetta-pMM064-pMM065 ON culture
inoculate 5 ml LB + Amp + IPTG(10 mM) with BAP1-pMM064
ON cultures opaque, but colorless
according to <bib id="pmid23053349"/>, indigoidine is extremely unstable, they did not observe any color above

30°C

inoculate 5 ml LB + Amp + Kan with 1 ml from Rosetta-pMM064-pMM065 ON culture
inoculate 5 ml LB + Amp with 1 ml from BAP1-pMM064 ON culture
when cultures reach opaqueness: add 1 mM IPTG, shake at 25-28°C
liquid cultures opaque, but colorless
indigoidine extremely unstable, but incubator has transparent door, incubator room well lit
prepare Amp+IPTG plate, plate BAP1-pMM064 from ON culture, leave at RT in drawer -> dark
inoculate 5 ml LB + Amp + Kan with 100 µl from Rosetta-pMM064-pMM065 ON culture
inoculate 5 ml LB + Amp with 100 µl from BAP1-pMM064 ON culture
when cultures reach opaqueness: add 1mM IPTG, wrap in tin foil (-> dark), shake at 25-28°C
no coloration of liquid cultures
white colonies on plate

Results and Discussion

Cells don't show a blue phenotype. We received the E. coli BAP1-strain which is commonly used for heterologuos

expression of NRPS. It carries sfp, a 4'-Phosphopanthetheinyl-Transferase from Bacillus subtilis to be able to

activate NRPS PCP-domains (i.e. T-Domains)[Pfeifer 2001]. Transformation with pMM64 (bpsA) should result in

indigoidine production since sfp was previously shown to activate indigoidine synthetases [Yu 2013].

Lab book

We received the E. coli BAP1-strain which is commonly used for heterologuos expression of NRPS. It carries sfp, a

4'-Phosphopanthetheinyl-Transferase from Bacillus subtilis to be able to activate NRPS PCP-domains (i.e. T-

Domains)[Pfeifer 2001]. Transformation with pMM64 (bpsA) should result in indigoidine production since sfp was

previously shown to activate indigoidine synthetases [Yu 2013].

Indigoidine production with pMM-plasmids III (Ilia)

co-transform BAP1 with 84.5 ng pMM64 and 225 ng pMM65, plate on Amp + Kan + IPTG

plate

inoculate 4 ml LB + Amp with BAP1-pMM64, grow at 37°C

Gel electrophoresis of the pMM64-pMM65 digestion. Lane 1: NEB 2-log ladder; lane 2: Undigested pMM64; lane 3: Digestion of pMM64 with EcoRI+NotI; lane 4: Digestion of pMM65 with Pst+XhoI

only white colonies on Amp + Kan + IPTG plate
make miniPrep of BAP1-pMM64 ON culture -> 19 ng / µl
digest 190 ng pMM64 with EcoRI+NotI (10 µl of miniPrep, 30 µl total volume), digest 450 ng pMM65 with PstI+XhoI

(30 µl total volume) => expect: 2 bands at 3.5 and 4 kb for pMM64, bands at 3.3 and 0.8 kb for pMM65

pMM64 shows fragments at 4 kb and 5 kb => wrong plasmid
pMM64 shows one weak band at 4 kb => might be right, but NanoDrop concentration measurement wrong
add 30 µl H₂O to original Fussenegger filter paper of pMM64
transform BAP1 with 2 µl pMM64 from original filter paper, plate on Amp + IPTG, grow

at 37°C

transform TOP10 with 2 µl pMM64 from original filter paper, plate on Amp, grow at

37°C

Gel electrophoresis of the pMM64 digestion. Lane 1: NEB 2-log ladder; lane 2: Undigested pMM64; lane 3: Digestion of pMM64 with EcoRI+NotI

White BAP1-pMM64 colonies
inoculate 4 ml LB + Amp with TOP10-pMM64 from plate
miniPrep: 15.2 ng / µl in 27.5 µl
digest 152 ng pMM64 (10 µl of miniPrep) with EcoRI+NotI (total volume 30 µl)
2 bands at 4.5 and 6 kb => wrong plasmid

Gel electrophoresis of the pMM64-pMM65 digestion. Lane 1: NEB 2-log ladder; lane 2: Digestion of pMM64 with EcoRI+HindIII; lane 3: Digestion of pMM65 with EcoRI+HindIII

inoculate 4 ml LB + Amp with TOP10-pMM64 from plate
miniPrep: 14.6 ng / µl in 27.5 µl
digest 175.2 ng (12 µl from miniPrep) pMM64 with EcoRI+HindIII (30 µl total volume, NEB buffer 2 + BSA)
digest 7 µl pMM65 with EcoRI+HindIII (30 µl total volume, NEB buffer 2 + BSA)
pMM64: 2 bands at 4.5 and 6 kb => wrong plasmid
pMM65: 1 band at 4 kb => might be right
add 30 µl H₂O to original Fussenegger filter paper of pMM65
transform TOP10 with 10 µl pMM65 from original filter paper, plate on Kan, grow at

37°C

inoculate 5 ml LB + Kan with TOP10-pMM65
miniPrep of pMM65 -> 23 ng / µl
prepare TOP10-pMM65 glycerol stock

Lab book

Indigoidine production with pMM-plasmids IV (Ilia)

inoculate 5 ml LB + Kan with TOP10-pMM65
miniPrep of pMM65 -> 23 ng / µl
prepare TOP10-pMM65 glycerol stock

run PCR for T and TE domain of bpsA for pMM64 validation and of svp for pMM65 validation:

PCR for pMM64/65 validation (17-06-13); Ladder was missing, probably taken loading buffer by mistake; run at 100 V, 0.8 % gel (TAE); wanted amplicon size are: * bpsA_T: ~250 bp * bpsA_TE: ~850 bp * svp: ~800 bp

- template each:
  - 5 ng of miniPrep (from 2013-06-15)
  - picked colony from transformed TOP10
  - water as control
- polymerase: Taq (validation PCR)

used cycler protocol for svp and T domain of bpsA:

Cycles	temperature [°C]	Time [s]
1	95	180
12	95	30
	71 (incr. down with 0.5 °C)	30
	95	30
18	95	30
	65	30
	95	60
1	72	600
1	4	inf

used cycler protocol for TE domain of bpsA:

Cycles	temperature [°C]	Time [s]
1	95	180
12	95	30
	63 (incr. down with 0.5 °C)	30
	95	30
18	95	30
	57	30
	95	60
1	72	600
1	4	inf

retransformation from filter paper provided bei Fussenegger group
- TOP10
- with 10 µl pMM64 from original filter paper, plate on Amp, grow at 37°C
- with 10 µl pMM65 from original filter paper, plate on Kan, grow at 37°C
prepare TB medium for ON (for comparement with LB)
colony PCR for plated pMM64 and pMM65 strains from 2013-06-16
prepare combinatorial experiment with gibson cloning or CPEC (polysytronic expression of bpsA and svp in TOP10)
prepare primer for bpsA only plasmid for BAP1 validation

===Results and Discussion===

Lab book

We want to assemble pKH1, which is a pSB1C3-derived plasmid containing pSB1C3-lacPromotor-BBa_B0034-bpsA(pMM64)-

BBa_B0034-svp(pMM65). Single fragments are amplified and cloned using a Gibson approach. We received Streptomyces lavendulae lavendulae since S. lavendulae ATCC11924 has been shown to carry bpsA

[Takahashi 2007].

Fragment amplification for pKH1 (Konrad)

prepare TB medium for ON (for comparement with LB)
colony PCR for plated pMM64 and pMM65 strains from 2013-06-16
prepare combinatorial experiment with gibson cloning or CPEC (polysytronic expression of bpsA and svp in TOP10)
prepare primer for bpsA only plasmid for BAP1 validation

0.4 µl template
2x 5 µl Primer (1:10 dilution)
25 µl Phusion MM
14.6 µl H2O

fragment	primer	template (DNA)	annealing temp (X1;X2) [°C]	elongation time (Y) [s]
f1: bpsA (all)	(NI01,NI06)	pMM64	66 (30x, no touch down)	135
f2: bpsA (AOxA)	(NI01,NI02)		68;65	60
f3: bpsA (T)	(NI03,NI04)
f4: bpsA (TE)	(NI05,NI06)
f5: bpsA (T-TE)	(NI03,NI06)
f6: svp	(NI07,NI08)	pMM65
f7: pSB1C3 (linear.)	(NI09,NI10)	pSB1C3 with J04450 (pJM03)	66;61	90

Cycles	temperature [°C]	Time [s]
1	98	30
10	98	1
	X1 (incr. down with 0.5 °C)	5
	72	Y
20	98	1
	X2	5
	72	Y
1	72	240
1	4	inf

gel purification

Assembly of pKH1 and Transformation (Konrad)

Gibson approach

molecular ratio = backbone : inserts = 1 : 3
partslength: bpsA=3.8 kbp; svp=0.8 kbp; pSB1C3=2.4 kbp

fragment	volume [µl]	DNA amount [ng]
f2: bpsA (AOxA)	3	126
f5: bpsA (T-TE)	1.5	60
f6: svp	1.5	109
f7: pSB1C3 (linear.)	2	51
total	8
	2 µl	H2O
	10 µl	Gibson MM

CPEC approach

Two different approaches were used. In the first approach 3 fragments (backbone (pSB1C3 based), bpsA and svp) were

used for assembly while for the second approach 4 different fragments (Backbone, AOxA, T-TE, svp) were used (like

for the Gibson mix). The amount of used template was calculated for backbone : insert ratio of 1:1 and for insert

fragment ratio at equimolar amount.

3 fragment mix
- 3 µl template (f7:backbone)
- 8 µl template (f1:bpsA)
- 0.3 µl template (f6:svp)
- 12.5 µl Phusion MM
- 1.2 µl H2O

4 fragment mix
- 3 µl template (f7:backbone)
- 1.7 µl template (f2:AOxA)
- 1.5 µl template (f5:T-TE)
- 0.3 µl template (f6:svp)
- 12.5 µl Phusion MM
- 6 µl H2O

The used cycler program was carried out similar to CPEC paper for multipart assembly but I forgot to note down the

exact parameters. (Shame on me...)

Transformation

into TOP10
4 plates (1:1 and 1:4 of Gibson mix; 1x of each CPEC mix)

Colony PCR

colony PCR of construct assembeled by Gibson; wanted amplicon size is around 1 kbp

analytical digestion of selected clones

validation of transformed BAP1 with Fussenegger plasmids

fusion PCR

Gibson Assembly of bpsA + svp insert

PCR for colony validation

IPTG induction, titration

IPTG induction, titration RESULT

Fussenegger plasmids in BAP1 under various conditions. BAP1 without any Plasmid, BAP1 transformed only with the bpsA carrying plasmid pMM64 and BAP1 transformed with the bpsA carrying plasmid pMM64 and svp carrying pMM65 were induced with four different IPTG concentration ranging from 100 µM to 10 mM at two different temperatures (18 °C and 24°C) for around 24 h.

CPEC and transformation

IPTG induction, titration RESULT

2 ml of samples from induction experiement from 2013-06-22 were centrifugated down and then visually compared. Like it can be seen, indigoidine is exported into the supernatant.

Streptomyces cultivation (Ralf)

media for streptomyces culture

YEME medium (Kieser et al. 2000)
- 10 g Glucose
- 3 g Yeast extract
- 5 g Bacteriological Peptone (I used Pankreatic Digest of Casein)
- 3 g Malt Extract
- 340 g Sucrose
- ad 1 L water

DSMZ medium 65 GYM streptomyces medium
- 4 g Glucose
- 4 g Yeast extract
- 10 g Malt extract
- (2 g CaCO3 for plates)
- (12 g Agar for plates)
- ad 1 L water

Results and Discussion

BAP1+pMM64+pMM65 eventually produced indigoidine. We will now focus on assembling our own constructs starting with

pKH1. Streptomyces cultures look like yeast and have a strong smell as in the forest.

Lab book

In parallel to the experiments with the pMM-plasmids (Konrad) we start to get the native bpsA from Streptomyces

lavendulae lavendulae DSMXXXX (Ralf).

Indigoidine production with pKH1 (Konrad)

colony PCR

transformation

analytic digestion

fragment amplification

PCR for bpsA and backbone amplification and Phusion polymerase validation (lanes to the right side of ladder; white stripe on the right is not a band); run at 135 V, 0.8 % gel (TAE); wanted amplicon size are: (bpsA: ~3.8 kbp; linearized pSB1C3: ~2.4 kbp)

==> for two fragments with our Phusion MM and Phusion MM of SYNtheSYS (old, don't know if functional...)

0.4 µl template
2x 0.5 µl Primer
25 µl Phusion MM
14.6 µl H2O

fragment	primer	template (DNA)	annealing temp (X1;X2) [°C]	elongation time (Y) [s]
f1: bpsA (all)	(NI01,NI06)	pMM64	65;66	120
f7: pSB1C3 (linear.)	(NI09,NI10)	pSB1C3 with J04450 (pJM03)	66;61	75

Cycles	temperature [°C]	Time [s]
1	98	30
10	98	5
	X1 (incr. down with 0.5 °C)	15
	72	Y
20	98	5
	X2	15
	72	Y
1	72	360
1	4	inf

RESULT: no PCR product for both amplicons, maybe due to wrong cycle conditions

construction of pSB1C3-bpsA-svp pRB1

Streptomyces lavendulae lavendulae DSMZXXXX was cultivated in GYM medium from freeze dried cell pellet at 28 °C and 170 rpm. cultivation of S. lavendulae lavendulae

freeze dried cell pellet in 5 ml medium 65
3x 200 ul in 3 mL medium 65 and YEME, respectively. 28 °C 200 rpm (3 p.m.)
3x agar plate medium 65, 50/75/100 ul inoculation; 28 °C (7 p.m.)
prepare YEME agar for next day

PCR with Gibson Primers RB05-RB10

pSB1C3 backbone from PCR product 50 uL Phusion Flash; 05 uM Primer, 25 uL Master mix, 19,4 uL water, 0,6 uL pSB1C3

Cycles	temperature [°C]	Time [s]
1	98	10
30	98	1
30	72	40
1	72	420
1	4	inf Gibson assembly and

BpsA colony PCR 50 ul Phusion Flash; 0,5 uM Primer, 25 uL Master Mix, 20 uL colony with water
colony was picked and held into liquid nitrogen/ warm water repeatedly

Cycles	temperature [°C]	Time [s]
1	98	11
1	65	5
1	72	60
30	98	1
	72	60
	1	72	300
1	4	inf

pMM65 PCR for svp 50 ul Phusion Flash; 0,5 uM Primer, 25 uL Master mix, 15 uL water, 5 uL 23 ng/ul pMM65

Cycles	temperature [°C]	Time [s]
1	98	11
1	65	5
1	72	15
30	98	1
30	72	15
1	72	300
1	4	inf

Agarose Gel

pSB1C3 backbone
- weak band on gel < 50 ng/ 17 ul
- Troubleshooting
  - two step PCR -> try annealing step with 66 °C annealing temp (NEB Tm Calculator Phusion)
  - circular template
  - template sequence?
bpsA
- cells not lysed? DNA damaged (nitrogen)?
svp

#2 PCR pSB1C3 RB09/10

8 ul water
10 ul Phusion Flash HF MM
je 0,5 ul Primer 100 uM
1 ul pSB1C3 1:5

Cycles	temperature [°C]	Time [s]
1	98	10
30	98	1
	62	5
	72	45
1	72	180
1	4	inf

PCR svp RB07/08 from pMM65 23 ng/ ul circular

8 ul water
10 ul Phusion Flash HF MM
je 0,5 ul Primer 100 uM
1 ul pMM65 1:10

Cycles	temperature [°C]	Time [s]
1	98	15
30	98	1
	62	5
	72	15
1	72	300
1	4	inf

PCR bpsA RB05/06 from S. lavendulae

Streptomyces was washed 3x in H20 dest; 2 "colonies" in 1 uL
8 ul water
10 ul Phusion Flash HF MM
je 0,5 ul Primer 100 uM
1 ul Streptomyces culture

Cycles	temperature [°C]	Time [s]
1	98	180
30	98	1
	62	5
	72	60
1	72	300
1	4	inf

0,8 % Agarose Gel

pSB1C3 worked well with 20-50 ng/ ul
BpsA
- annealing temp still too high?
- cell lysate destroys master mix? -> nitrogen/ boil before PCR, use liquid phase
- cells not lysed? vortex with glas beads

#3 desperation PCR

PCR	water	Phusion Flash HF MM	Primer 100 uM	DMSO	template	template
Ia	6	10	RB05, RB06 à 0,5 ul	1	2	S. lav, medium 65, nitrogen shock, glass beads vortex, 98 °C with water bidest; liquid phase
Ib	6	10	RB05, RB06 à 0,5 ul	0	3	S. lav, medium 65, nitrogen shock, glass beads vortex, 98 °C with water bidest; liquid phase
II	6	10	RB05, RB06 à 0,5 ul	0	3	S. lav, medium 65, glass beads vortex, 98 °C with water bidest; liquid phase
III	6	10	RB05, RB06 à 0,5 ul	0	3	S. lav, medium 65, nitrogen shock, 98 °C with water bidest; liquid phase
IVa	6	10	RB05, RB06 à 0,5 ul	1	2	S. lav, medium 65, 98 °C with water bidest; liquid phase
IVb	6	10	RB05, RB06 à 0,5 ul	0	3	S. lav, medium 65, 98 °C with water bidest; liquid phase
V	6	10	RB05, RB06 à 0,5 ul	0	3	S. lav, medium 65, nitrogen shock, glass beads vortex; pellet
VI	6	10	RB05, RB06 à 0,5 ul	0	3	S. lav, YEME medium, nitrogen shock, glass beads vortex; pellet/ liquid phase
VII	6	10	RB05, RB06 à 0,5 ul	0	3	S. lav, YEME medium, nitrogen shock, 98 °C with water bidest; liquid phase
VIII	7	10	RB07, RB08 à 0,5 ul	1	1	pMM65 2,3 ng/ ul, trace RB05
IX	6	10	RB07, RB08 à 0,5 ul	0	1	pMM65 2,3 ng/ ul, should be 8 ul water

Cycles	temperature [°C]	Time [s]
1	98	120
2	98	1
	40	5
	72	70
2	98	1
	45	5
	72	70
5	98	1
	50	5
	72	70
5	98	1
	55	5
	72	70
15	98	1
	65	5
	72	70
1	72	300
1	4	inf

Desperation Gel

0,8 % Agarose, 100 V, 60 min.

band around 200 bp? unspecific product due to PCR conditions, but then why no product?
VI glowing pocket -> sucrose in YEME medium? -> PCR purification and new gel -> still nothing

Phusion Flash HF has proofreading activity; so mutations may stop elongation -> try intron iTaq and design new

primers without mutations.

PCR svp from NI7/8 PCR product and pMM65, respectively; svp with NI7/8 as control; intron iTaq 2x Master mix.

PCR	iTaq 2x Master Mix	template	Primer 10 uM	water
A	10 ul	pMM65 2 ul 2,3 ng/ ul	1 ul RB07 and RB08	6 ul
B	10 ul	pMM65 2 ul 2,3 ng/ ul	2 ul RB07 and RB08	4 ul
C	10 ul	svp 2 ul 7,2 ng/ ul	1 ul RB07 and RB08	6 ul
D	10 ul	svp 2 ul 7,2 ng/ ul	2 ul RB07 and RB08	4 ul
E	10 ul	svp 1 ul 7,2 ng/ ul	1 ul NI07 and NI08	8 ul

Cycles	temperature [°C]	Time [s]
1	94	120
35	94	20
	60	10
	70	60
1	72	240
1	4	inf

Gel 0,8 % 100 V 60 min

nothing
- intron iTaq maybe doesn't work so well since control isn't working; or standard protocol is bad.
- run PCR with other Taq Master Mix
iTaq with Slav colony YEME

Tube	Master Mix	MM 2x [ul]	Primer 100 mM [ul]	template	template [ul]
A:svp RB7/8	Fermentas Taq PCR Master Mix 2x	25	7,5	svp 7,2 ng/ ul	10
B:svp RB7/8	Fermentas Taq PCR Master Mix 2x	25	7,5	svp 7,2 ng/ ul	10
C:pMM65 RB7/8	Fermentas Taq PCR Master Mix 2x	25	7,5	pMM65 2,3 ng/ ul	10
D:pMM65 RB7/8	Fermentas Taq PCR Master Mix 2x	25	7,5	pMM65 2,3 ng/ ul	10
E:pMM65 NI7/8	Fermentas Taq PCR Master Mix 2x	25	7,5	pMM65 2,3 ng/ ul	10
F:svp RB7/8	Finnzymes Taq PCR Master Mix 2x	25	7,5	svp 7,2 ng/ ul	10
G:svp RB7/8	Finnzymes Taq PCR Master Mix 2x	25	7,5	svp 7,2 ng/ ul	10
H:pMM65 RB7/8	Finnzymes Taq PCR Master Mix 2x	25	7,5	pMM65 2,3 ng/ ul	10
I:pMM65 RB7/8	Finnzymes Taq PCR Master Mix 2x	25	7,5	pMM65 2,3 ng/ ul	10
J:pMM65 NI7/8	Finnzymes Taq PCR Master Mix 2x	25	7,5	pMM65 2,3 ng/ ul	10
K:svp RB7/8	NEB OneTaq Quick Load PCR Master Mix 2x	25	7,5	svp 7,2 ng/ ul	10
L:svp RB7/8	NEB OneTaq Quick Load PCR Master Mix 2x	25	7,5	svp 7,2 ng/ ul	10
M:pMM65 RB7/8	NEB OneTaq Quick Load PCR Master Mix 2x	25	7,5	pMM65 2,3 ng/ ul	10
N:pMM65 RB7/8	NEB OneTaq Quick Load PCR Master Mix 2x	25	7,5	pMM65 2,3 ng/ ul	10
O:pMM65 NI7/8	NEB OneTaq Quick Load PCR Master Mix 2x	25	7,5	pMM65 2,3 ng/ ul	10

500px|2-log; A-E; 2-log; F-J

2-log; K-O; 2-log; Slav IVb; VI; V; VII

gel analysis
- svp worked well as template for RB07/08, but lots of wrong product around 100-150 bp. Maybe primers bind to each other, folding results in other preferred sequence or gibson overlap has big affinity though alignments didn't show that
- pMM65 amplification only with NEB OneTaq
- no product with NI07/08 primers...because they are for TE-domain of BpsA. Ni09/10 would have been the right choice
- for further runs, use OneTaq and Fermentas Taq
- was svp amplified or is it just template?
- Again glowing pocket in Slav VI
- huge amount of primers was used in al reactions

PCR with Phusion Flash HF from PCR products (yesterday with Taq-polymerases)

10 ul MM; Primer je 2 ul unv.; water 4 ul; template 2 ul

Cycles	temperature [°C]	Time [s]
1	98	10
30	98	1
	55	5
	72	30
1	72	300
1	4	inf

gel 9 ul 1x loading buffer, 1 ul pcr product

2-log / A / A-Phu Fl / F / F-Phu-Fl / K / K-Phu-Fl / svp 7,2 ng template

gel analysis
- no significant amplification of svp with Phusion Flash
- Taq PCR amplified template
- run different protocol
PCR Phusion flash taq-amplified svp 2
- 10 ul MM, 1 ul primer, 7 ul water, 1 ul water

Cycles	temperature [°C]	Time [s]
1	98	10
30	98	1
	65	5
	72	30
1	72	180
1	4	inf

Cycles	temperature [°C]	Time [s]
1	98	10
30	98	1
30	72	35
1	72	180
1	4	inf

gel with 9 ul 1x loading buffer and 1 ul pcr product

2-log / wrong / wrong / 3-step K / 3-step K-Phu-Fl / 2-step K / 2-step K-Phu-Fl / K template / 2-log

gel analysis
- 3-step worked better than 2-step
- primers might prime each other or the fragment
- it's not just too much primer because lane 4/5 are a lot brighter than 6/7 -> the 120 bp-band is an

amplification product. -> order S. verticillus and published primers from Takahashi and Sanchez -> order published Primers for sfp, entD (Lambalot), bpsA (Takahashi) -> order stuff for DNA isolation of Streptomyces ===Results and Discussion===

Lab book

Indigoidine production with pKH1 II (Konrad)

fragment amplification

==> f1: bpsA (all)

14.6 µl H2O
25 µl Phusion Flash
2x 0.5 µl Primer (NI01,NI06)
0.4 µl template pMM64

Cycles	temperature [°C]	Time [s]
1	98	10
30	98	1
	66	5
	72	120
1	72	420
1	4	inf

PCR for amplification of bpsA (all); lane 11: marker, lane 12 and 13: f1:BpsA (all), rest=see Del run at 135 V, 0.8 % gel (TAE); wanted amplicon size is: bpsA: ~3.8 kbp

Bands cut out of PCR (amplification of bpsA (all))

Gel purification (50µl): by accident did not take 3 but 1 Volume buffer QG

Labelling fragements: 1 I and 1 II (from different lanes)

fusion PCR

==> for fusion of both fragments bpsA and svp using Phusion Flash HF

19.8 µl H2O
25 µl Phusion Flash MM
2x 1.0 µl Primer (NI01,NI08)
2.6 µl template (f1:bpsA)
0.6 µl template as 1:10 dilution (f6:svp)

Cycles	temperature [°C]	Time [s]
1	98	10
30	98	1
30	72	70
1	72	180
1	4	inf

PCR for fusion of bpsA (all) and svp: lane 3 & 4, lane 6: marker, rest=see Del run at 135 V, 0.8 % gel (TAE); wanted amplicon size is: bpsA and svp: ~4.6 kbp

PCR for fusion of bpsA (all) and svp, cutted

Gel purification (50µl): by accident did not take 3 but 1 Volume buffer QG

Labelling fragments: 1 I and 1 II (from different lanes)

Amplification pSB1C3 (f7)

==> f7: pSB1C3 (linear.) with our Phusion MM and Phusion MM of SYNtheSYS (old, don't know if functional...)

PCR for amplification of pSB1C3: lane 10 & 11: our fusion MM, lane 12 & 13: Synthesys fusion MM, lane 1: marker, rest=see Del Rest, run at 135 V, 0.8 % gel (TAE); wanted amplicon size is: 2,4 kbp

(add items in this order)

14.6 µl H2O
25 µl Phusion MM
2x 0.5 µl Primer (NI09,NI10)
0.4 µl template (pSB1C3 with J04450 (pJM03))

Cycles	temperature [°C]	Time [s]
1	98	30
30	98	10
30	72	90
1	72	420
1	4	inf

-->Hot start

RESULT: no product neither with Synthesys fusion nor our fusion

==> f7: pSB1C3 (linear.) with Fusion Flash

(add items in this order)

14.6 µl H2O
25 µl Phusion Flash
2x 0.5 µl Primer (NI09,NI10)
0.4 µl template (pSB1C3 with J04450 (pJM03))

Cycles	temperature [°C]	Time [s]
1	98	10
30	98	1
30	72	40
1	72	420
1	4	inf

PCR for amplification of pSB1C3: lane 3 & 4, lane 1=marker, rest=see Indigoidine Streptomyces, run at 135 V, 0.8 % gel (TAE); wanted amplicon size is: bpsA: ~3.8 kbp

Bands cut out of PCR (amplification of pSB1C3)

fusion PCR

==> for fusion of both fragments bpsA and svp using Phusion Flash HF

19.8 µl H2O
25 µl Phusion Flash MM
2x 1.0 µl Primer (NI09,NI10)
5 µl template (f1:bpsA I,03.07.13)
0.6 µl template as 1:10 dilution (f6:svp)

--> wrong primers, should have taken NI01 and NI08
--> more than 50µl as not enough bpsA template was left

Cycles	temperature [°C]	Time [s]
1	98	10
30	98	1
30	72	70
1	72	180
1	4	inf

Re-PCR of fusion (bpsA+svp)

==> for amplification of fusion fusion product 03.07 (bpsA + svp) using Phusion Flash HF

18 µl H2O
25 µl Phusion Flash MM
2x 1.0 µl Primer (NI09,NI10)
5 µl template (fusion bpsA+svp,03.07.13)

--> wrong primers, should have taken NI01 and NI08

Cycles	temperature [°C]	Time [s]
1	98	10
30	98	1
30	72	70
1	72	180
1	4	inf

Re-PCR of fusion (bpsA+svp)

==> for amplification of fusion fusion product 03.07 (bpsA + svp) using Phusion Flash HF

19 µl H2O
25 µl Phusion Flash MM
2x 0.5 µl Primer (NI01,NI08)
5 µl template (fusion bpsA+svp,03.07.13)

Cycles	temperature [°C]	Time [s]
1	98	10
30	98	1
30	72	70
1	72	180
1	4	inf

Preparations

prepare liquid culture of Rosetta + pMM64 + pMM65 in 2 ml LB with Amp + Kan for plate oculation
prepare liquid cultures of TOP10 + pMM64/65 for minipreps: 3 ml TB + Amp/Kan
prepare liquid cultures for induction test: 3 ml LB + appropiate AB
- BAP1
- BAP1 + pMM64 (Amp)
- BAP1 + pMM64 + pMM65 (Amp + Kan)
- TOP10 + pJH1 (Kan)

Re-PCR of fusion (bpsA+svp)

Ralf

PCR from glycerol stock S. lavendulae with Fermentas; Thermoscientific and NEB Taq 50 ul

MM 25 ul, 7,5 ul primer RB7/8 100 mM, 5 ul water, 5 ul culture

Cycles	temperature [°C]	Time [s]
1	95	300
30	95	30
	50	40
	70	300
1	70	300
1	4	inf

S. verticillus and S lavendulae in 50 ml YEME medium for 46 h at 28 °C.
filamentous growth and spherical colonies in S. lavendulae flask after 1 day -> contamination?!
no growth in S. verticillus flask after 18 h
prepare plates

colony PCRs

	1 bpsA S. lav	2 svp S. vert	3 svp S. vert	4 entD MG1655	5 sfp BAP1
water	10	10	10	10	10
Phusion Flash MM	25	25	25	25	25
Primer	à 5 ul RB11/12	à 5 ul RB13/14	à 5 ul RB15/16	à 5 ul RB19/20	à 5 ul RB17/18
template	colony pick from plate	cell pellet liquid culture	cell pellet liquid culture	cell pellet glycerol stock\|\|colony from plate
	biometra	BioRAD test machine

colony PCRs 50 ul Phusion flash (25 ul + 5 ul Primer 10 mM + 10 ul water

bpsA - S. lav (Takahashi) RB11/12
svp - S. vert (Taka Primer) RB13/14
svp - S. vert (Sanchez) RB15/16
entD - MG1655 (Lambalot) RB19/20
sfp - BAP1 RB17/18

biometra

1	98	120
98	1
	65	5
	72	20/60
12	-

Gel

2-log; bpsA; svp 13/14; svp 15/16; entD; sfp

Gradient PCR with both Streptomyces under same conditions to validate PCR parameters. Phusion Flash HF 20 ul (10 ul MM; 2 ul Primer 10 mM; 2 uL template (cell pellet from GYM 48 h liquid culture; 6 ul

H2O)

1	98	120
30	98	1
	53-57-59-61-65	5
	72	60
12	-

Gel

===Results and Discussion===

Lab book

Indigoidine production pKH and pMM (Konrad)

IPTG Induction

Induction of 2 days old cultures with/-out 1 mM IPTG. Rosetta seems to produce indigoidine, even without induction (leaky exppression?).

Occulate 2 ml LB + appropiate AB with:

Rosetta + pMM64 + pMM65
BAP1
BAP1 + pMM64
BAP1 + pMM64 + pMM65

Rosetta was oculated from plate (06.07.), the BAP1 culture with 10 µl of ON from 06.07. These DC were incubated for

around 7 hours at 37 °C.

Old broth from 06.07 were induced with 1 mM IPTG at 24 °C for 2 hours. Rosetta strain becomes very blue, even

without IPTG induction.

fusion PCR (bpsA+svp)

based on PCA protocol: Used two main steps, first elongation of fragments (f1;f6) without any primers, than

enrichment of desired fusion product with fw primer of fragment 1 and rv primer of fragment 2.

PCA in order to gain bpsA and svp as one fragment fused together. The wanted fragment size is ~ 4.6 kbp, so the blue indicated bands were cut out.

Step 1:

13 µl H2O
25 µl Phusion Flash MM
2 µl template (f6:svp)
10 µl template (f1:bpsA from 4.07.)

(concentration of f1 from 4.07. unknown)

Step 2:

23.5 µl H2O
25 µl Phusion Flash MM
0.5 µl template (reaction from step 1)
2x 0.5 µl primer (NI:01; NI:08)

Step	Cycles	temperature [°C]	Time [min]
1	1	98	0:10
	5	98	0:01
		52	0:30
		72	0:20
	1	72	5:00
	1	12	inf
use only 0.5 µl of mixture for 2nd step
2	1	98	0:10
	30	98	0:01
	30	72	1:10
	1	72	5:00
	1	12	inf

Gel extraction of ~4.6 kbp bands cut out

Gibson assembly

molecular ratio = backbone : inserts = 1 : 3
partslength: bpsA=3.8 kbp; svp=0.8 kbp; pSB1C3=2.4 kbp

fragment	volume [µl]	DNA amount [ng]
f8: bpsA + svp	7.5	214
f7: pSB1C3 (linear.)	2.5	55
total	10
	0 µl	H2O
	10 µl	Gibson MM

transformation of TOP10 with 2 µl directly of GA reaction mix and 1:4 dilution respectively

additional

preparations of JM1 from 4.5 ml TOP10 ON culture (used a bit for transformation, gave rest to Johanna)
transformation of BAP1 with 88 ng (5 µl) JM1 (probably way to much DNA)
IPTG induction (1mM) experiment with Rosetta + pMM64 + pMM65, BAP1, BAP1 + pMM64, BAP1 + pMM64 + pMM65 (DC, was

in 4 °C fridge overnight)

- RESLUT: did not turn blue at all, may the IPTG be bad? (did not use the same as the day before)

CPEC Assembly

CPEC for pKH1 assembly: only insert seems visible, no backbone and CPEC product; run at 135 V, 0.8 % gel (TAE); wanted amplicon size is: 2,4 kbp

fragment	volume [µl]	DNA amount [ng]
f8: bpsA + svp	2.3	150
f7: pSB1C3 (linear.)	5.2	50
total	7.5
	2.5 µl	H2O
	10 µl	PhusionFlash MM
total	20

Cycles	temperature [°C]	Time [min]
1	98	0:10
3	98	0:01
	55	0:30
	72	2:00
1	72	5:00
1	12	inf

use 10 µl for gel picture, store 10 µl

PCR amplification f7 (pSB1C3)

PCR for amplification of pSB1C3: (left) lane 1 & 2: todays amplification, lane 3: Marker, lane 4: 2 µl of gel/nucleotide purified amplification of 4th of July, lane 5 & 6: delRest 11 - 12 with long and short primers (unknown order); (right) gel cut out run at 135 V, 0.8 % gel (TAE); wanted amplicon size is: 2,4 kbp

(add items in this order)

14.6 µl H2O
25 µl Phusion MM
2x 0.5 µl Primer (NI09,NI10)
0.4 µl template (pSB1C3 with J04450 (pJM03))

Cycles	temperature [°C]	Time [min]
1	98	0:10
30	98	0:01
30	72	0:40
1	72	5:00
1	12	inf

Ralf

new primers are there yippie

pSB1C3 RB21/22

2,5 ul template pSB1C3 1:5
25 ul Phusion Flash MM
5 ul Primer 10 mM
12,5 ul water

1	98	10
1	98	1
	61	5
	72	60
29	98	1
29	72	60
1	72	300
1	12	-

T1000 BIORAD

indC-Plum RB27/28

1 ul template cell pellet from DSMZ stem culture
25 ul Phusion Flash MM
5 ul Primer 10 mM
14 ul water

1	98	120
1	98	1
	57	5
	72	90
29	98	1
	65	5
	72	90
1	72	300
1	10	-

biometra

sfp-BAP1 RB35/36

1 ul template cell pellet from BAP1 liquid culture
25 ul Phusion Flash MM
5 ul Primer 10 mM
14 ul water

1	98	120
1	98	1
	63	5
	72	30
29	98	1
	65	5
	72	30
1	72	300
1	10	-

BioRAD 2-block

Electrophoresis 1 % Agarose 0,5 % TAE 100 V 60 min; PCR product 50 ul + 10 ul 6x loading dye 10 ul quickload 2-log; 2 ul PCR pSB1C3-indC-sfp; 29 ul 2x pSB1C3; 2x indC; 2x sfp

400px|1st run with long primers

400px|cut backbone for gel extraction

indC-Plum #2 Q5 50 ul 1, 2: RB37/38; 3: RB27/28

5 ul template liquid culture - pellet
25 ul Q5 MM
5 ul Primer 10 mM
10 ul water

cycles	temperature °C	time seconds
1	98	180
30	98	8
	56	10
	72	150
1	72	300
1	12	-

sfp BAP1 #2 Q5 50 ul a: RB35/36 b: RB43/44

template colony pick
25 ul Q5 MM
5 ul Primer 10 mM
15 ul water

cycles	temperature °C	time seconds
1	98	180
30	98	8
	62	10
	72	40
1	72	300
1	12	-

2-log (1 ug); (1 ul) 1 - 2 - 3 - a - b; (all) 1 - 3 - 3 - a - b - b

analysis

indC fragmet with short primers worked well -> gel extraction and re-PCR with long primers
sfp and indC long almost nothing -> improve conditions

sfp BAP1 #3 Q5 50 ul A: RB17/18; naka; 60 °C B: RB17/18; naka; 56 °C C: RB43/44; short; 60 °C D: RB43/44; short; 56 °C E: RB35/36; long; 60 °C F: RB35/36; long; 65 °C

template colony pick
25 ul Q5 MM
5 ul Primer 10 mM
15 ul water

cycles	temperature °C	time seconds
1	98	180
30	98	8
	56-60-65	10
	72	30
1	72	300
1	12	-

2-log (1 ug); A-F

analysis

B, C and E worked best -> gel extraction
re-pcr E

fragments for pRB3

rePCR from indC-Plum #2 with long primers
- 2x 50 ul Phusion Flash HF
- RB27/28 long primer for pRB3 5 ul 10 uM
- water 14.8 ul
- template 0.2 ul pcr gel extracted

biorad T100

cycles	temperature °C	time seconds
1	98	10
12	98	1
	td 57-53 -0.5	5
	72	70
18	98	1
	65	5
	72	70
1	72	300
1	12	-

rePCR from sfp-BAP1 #3 E with long primers
- 2x 50 ul Phusion Flash HF
- RB35/36 long primer for pRB3 5 ul 10 uM
- water 14.8 ul
- template 0.2 ul pcr gel extracted

biorad T100

cycles	temperature °C	time seconds
1	98	10
30	98	1
	65	5
	72	20
1	72	300
1	12	-

rePCR from pSB1C3 Hanna 1:5 with long primers
- 2x 50 ul Phusion Flash HF
- RB21/22 long primer for pRB3 5 ul 10 uM
- water 14.8 ul
- template 0.2 ul pcr gel extracted

cycles	temperature °C	time seconds
1	98	10
12	98	1
	td 64 -0.5	5
	72	70
23	98	1
	65	5
	72	70
1	72	300
1	12	-

2-log (1 ug); pSB1C3; indC; sfp

assembly pRB3

digest 25 ul (2.5 CutSmart 10x; 0.5 enzyme each; 21.5 DNA; 1 h at 37 °C)
- indC KpnI-HF and BamHI-HF
- sfp BamHI-HF and NheI-HF
- pSB1C3 NheI-HF and KpnI-HF

Ligation 1 20 ul (2.0 T4 buffer 10x; 1.0 T4; 10.0 indC; 4.0 sfp; 3.0 pSB1C3)
Ligation 2
- 2.0 T4 buffer 10x; 1.0 T4; 7.0 indC; 10.0
- 2.0 T4 buffer 10x; 1.0 T4; 10.0 product; 7.0 pSB1C3

CPEG 20 ul Phusion Flash HF (10 MM; 7 indC; 2 pSB1C3; 1 sfp)

biometra

cycles	temperature °C	time seconds
1	98	10
10	98	1
	3grad. 50 +5	5
	72	70
1	72	180
1	12	-

Transformation TOP10
- CPEG 50 5 ul
- CPEG 55 5 ul
- CPEG 55 plus 20 min T4 ligase 10 ul (5 ul + 5 ul ligation master mix)
- CPEG 60 5 ul
- Lig1 2 ul
- Lig1 4 ul
- Lig2 4 ul

TOP10-pRB3-CPEG50-5ul	TOP10-pRB3-CPEG55-5ul	TOP10-pRB3-CPEG55lig-5ul	TOP10-pRB3-CPEG60-5ul
TOP10-pRB3-lig1-2ul	TOP10-pRB3-lig1-4ul	TOP10-pRB3-lig2-4ul

-> order primers for the other PPTases and indigoidine synthetases (set RB21-44 complete)

===Results and Discussion===

Lab book

Indigoidine production Konrad (Konrad)

PCR for amplification of pSB1C3: (left) first analytical gel; (right) second analytical gel. f7 was twice eluted after gel extraction, the second run was 30 min in 15 µl H₂O at 50 °Ca before centrifugation; run at 100 V, 0.8 % gel (TAE);

gel extraction of yesterdays fragment f7:pSB1C3
analytical gel (small, half size): (M: 3 µl NEB log2, 2 µl f7 + 2 µl 1x loading dye)
==> [f7] = 3 ng/µl
2nd analytical gel (small, half size): (M: 3 µl NEB log2, (2 µl f1:4.07., 2 µl f7:14.07., 2 µl f7:14.07.-2nd

eluation, 2 µl delRest-f6-9) + 8 µl 1x loading dye)
==> [f1:4.07.] = 18 ng/µl, [f7:14.07.] = 5 ng/µl,

[f7:14.07.-2nd] = 2 ng/µl,[delRest-f] = 5 ng/µl

fusion PCR (bpsA+svp)

based on PCA protocol: Used two main steps, first elongation of fragments (f1;f7) without any primers, than

enrichment of desired fusion product with fw primer of fragment 1 and rv primer of fragment 2.

PCA in order to gain bpsA and pSB1C3 as one fragment fused together. The wanted fragment size is ~ 6.2 kbp.

Step 1:

10 µl Phusion Flash MM
6.3 µl template (f7:pSB1C3)
3.7 µl template (f1:bpsA from 4.07.)

Step 2:

20.0 µl H₂O
25 µl Phusion Flash MM
4.0 µl template (reaction from step 1)
2x 0.5 µl primer (NI:06; NI:09)

Step	Cycles	temperature [°C]	Time [min]
1	1	98	0:10
	5	98	0:01
		52	0:30
		72	0:20
	1	72	5:00
	1	12	inf
use only 4.0 µl of mixture for 2nd step
2	1	98	0:10
	30	98	0:01
	30	72	1:10
	1	72	5:00
	1	12	inf

Preparation for electroporation

analytical gel of purified Gibson assembly and delRest;run at 100 V, 0.8 % gel (TAE);

Gibsonassembly purified with Nucleotide Removal Kit
analytical gel (small, half size): (M: 2.5 µl NEB log2)
==> [delRest(short)] = 6-7 ng/µl, [delRest(long)] = 4

ng/µl, [GA] = 3 ng/µl

Validation of Gibson assembly

Gibson assembly. The wanted fragment size is ~ 7.0 kbp.;run at 100 V, 0.8 % gel (TAE);

put 10 µl on gel.
electroporation with left 4 µl

fusion PCR (bpsA+svp)

PCA in order to gain bpsA and pSB1C3 as one fragment fused together. The wanted fragment size is ~ 6.2 kbp.;run at 135 V, 0.8 % gel (TAE);

Redo 2nd step with 1st step result of the 13th of July

Step 2:

23.5 µl H₂O
25 µl Phusion Flash MM
0.5 µl template (reaction from step 1)
2x 0.5 µl primer (NI:06; NI:09)

Step	Cycles	temperature [°C]	Time [min]
2	1	98	0:10
	30	98	0:01
	30	72	1:10
	1	72	5:00
	1	12	inf

soil sample preparation

experimental CPEC assembly with left mix from day before

Analytical gel for verification of CPE success.

File:Heidelberg_20130719-CPEC.png

PCR of bpsA and svp with new Primers

PCR amplification of svp with new Primers.

PCR amplification of bpsA with new Primers.

gel extraction of new bpsA and svp fragments

Analytical gel bpsA and svp gel extraction.

Gibson Assembly
CPEC

Analytical gel for verification of Gibson assembly and CPEC success.

Trafo

Colony PCR of electroporation with GA

Colony PCR for pKH1 validation. The T-TE domain of bpsA was used as amplicon (size: 1.1 kbp) as well as svp-part of backbone (similar size of 1.1 kbp). Every lane represents one PCR reaction with 1 picked colony each. Except for colony 1 only black colonies were picked.

Colony PCR of trafos of day before

Colony PCR for pKH1 und pKH2 validation. The T domain of bpsA was used as amplicon (size: 300 bp). Every lane represents one PCR reaction with 3 picked colony each.

Heat-shock competent Rosetta
Minipreparation of 2 ml ON culture of one picked black colony of GA plate (electroporation)

Ralf

PCR PPTases #1 Phusion Flash HF 50 ul (long) 20 ul (short); colony pick/0.2 ul pcr template; primer 2/5 ul 10 uM

MG1655 pick RB33/34 and RB 41/42
S. vert pick liquid RB29/30 and RB 39/40
pMM65 pcr RB25/26

biorad T100

cycles	temperature °C	time seconds
1	98	180/10
12	98	1
	td 68 -0.5	5
	72	20
18	98	1
	65	5
	72	20
1	72	180
1	12	-

PCR bpsA64 Phusion Flash HF 50 ul (long); 0.2 ul pcr template; primer 5 ul 10 uM

pMM64 Fussenegger miniprep RB23/24

biometra

cycles	temperature °C	time seconds
1	98	10
12	98	1
	td 58 -0.5	5
	72	70
18	98	1
	65	5
	72	70
1	72	300
1	12	-

bpsA; entD-l; svpV-l; svpF-l; entD-s; svpV-s

PCR PPTases #3 Phusion Flash HF 50 ul (25 MM; 5/5 primer; 14.5 water; 0.5 template)

1: entD from PPTases #1 gel ex w/ RB33/34 long
2: svpV from ??? w/ RB29/30 long
3: svpF from PPTases #1 gel ex w/ RB25/26 long

cycles	temperature °C	time seconds
1	98	10
30	98	1
	65	5
	72	20
1	72	180
1	12	-

1 ug 2-log; entDlong; svpVlong; svpFlong

analysis

entD and svpF -> gel extraction
svpV is too small -> wrong product. new colony PCR with short primers

Konrad PCR svpV #3

cycles	temperature °C	time seconds
1	98	180
30	98	1
	3grad. 65-62-57	5
	72	20
1	72	180
1	10	-

2-log; RB13/14 65°C; RB39/40 62 °C; RB 39/40 57 °C

analysis

hmm

PCR svpV #4 20 ul Phusion Flash HF (10 MM; 9.4 water; 0.2 ul primer 100 uM; 0.2 cell pellet) conditions as in Jul 12th; RB13/14 biorad T100

cycles	temperature °C	time seconds
1	98	120
30	98	1
	65	5
	72	20
1	72	300
1	12	-

2-log; Hanna; svpV RB13/14

rePCR svpV #5 from #4 50 ul (25 MM; 14 water; 0.5 primer 100 uM; 0.2 template)

1: RB29/30 from short RB13/14
2: RB29/30 from short RB 39/40

biorad T100

cycles	temperature °C	time seconds
1	98	10
10	98	1
	td 65 -0.5	5
	72	20
25	98	1
	65	5
	72	20
1	72	300
1	12	-

2-log; from taka; from RB39/40

-> gel extraction; CPEG assembly

plasmid name	backbone	cutting site	indigoidine-Synthetase	cutting site	PPTase	cutting site	primers
pRB3	pSB1C3-BBa_B0034	KpnI	P.lum indC	BamHI	Bsub sfp	NheI	RB21/22-27/28-35/36
pRB4	pSB1C3-BBa_B0034	KpnI	P.lum indC	BamHI	Svert svp	NheI	RB21/22-27/28-29/30
pRB5	pSB1C3-BBa_B0034	KpnI	P.lum indC	BamHI	Svert svp65	NheI	RB21/22-27/28-25/26
pRB6	pSB1C3-BBa_B0034	KpnI	P.lum indC	BamHI	Eco entD	NheI	RB21/22-27/28-33/34
pRB7	pSB1C3-BBa_B0034	KpnI	S.lav bpsA64	BamHI	Bsub sfp	NheI	RB21/22-23/24-35/36
pRB8	pSB1C3-BBa_B0034	KpnI	S.lav bpsA64	BamHI	Svert svp	NheI	RB21/22-23/24-29/30
pRB9	pSB1C3-BBa_B0034	KpnI	S.lav bpsA64	BamHI	Svert svp65	NheI	RB21/22-23/24-25/26
pRB10	pSB1C3-BBa_B0034	KpnI	S.lav bpsA64	BamHI	Eco entD	NheI	RB21/22-23/24-33/34

CPEG assembly 20 ul Phusion Flash HF (10 MM; 2 pSB1C3; 1.5 PPTase; 6 ind synthetase) biometra

cycles	temperature °C	time seconds
1	98	10
10	98	1
	55	5
	72	70
1	72	180
1	12	-

Transformation 10 pm; 80 ul +/- 20 ul; 5 ul CPEG pcr product

medium LB: FG 2013Jul19
plates LB+Cm: FG 2013Jul22
TOP10 from stock -80 °C

Results and Discussion===

Lab book

===Konrad

validation of ccdB constructs

MP of pKH2(ccdB) constructs from independent colonies: 3rd and 4th were used according to colony PCR result
- (elution in 30 µl): col3=170 ng/µl ; col4=145 ng/µl
testtrafo in ccdB resistent Survival, TOP10 and DH5alpha cells (from Dominik) of constructs pKH2, pKH2(ccdB)-

col3, pKH2(ccdB)-col4

- transformation with 20 ng
- plated 30 µl of cell suspension on 3 partiated plates for each constructs

Construction of ccdb-constructs pRB11-13

pRB11, pRB12 (bpsA+svpF) and pRB13 (indC+sfp) carry the ccdb gene with RBS instead of their native T-Domain so that

they don't produce the blue pigment, survive in OneShot ccdb survival cells and die when transformed to TOP10 or

DH5alpha. If the new T-Domain is successfully inserted, transformed cells survive in TOP10 and hopefully produce

the blue pigment.

amplification of CPEC fragments

NI primer in this case are for pRB11 and pRB12; KH primer for pRB13 templates are miniprepped plasmids

ccdb (NI13/14; KH1/2; 350 bp)
construct pKH1/2 and pRB3 without T-Domain (NI15/16; KH3/4; 7000 bp)
indC T-Domain as a control (NI17/18; KH5/6; 200 bp)
bpsA T-Domain as a control (NI19/20; KH7/8; 200 bp)

component	concentration	amount
Phusion Flash HF MM	2x	25 ul
Primer each	10 uM	5 ul
water		14.7 ul
template	>20 ng/ul	0.3 ul

NI13/14 in T100
cycles	temperature (°C)	time (s)
1	98	10
8	98	1
	64 ↓ 0.5	5
	72	10
22	98	1
	65	5
	72	10
1	72	60
1	12	-
KH1/2 in biometra
cycles	temperature (°C)	time (s)
1	98	10
8	98	1
	56 ↓ 0.5	5
	72	10
22	98	1
	65	5
	72	10
1	72	60
1	12	-
KH3/4 in T100
cycles	temperature (°C)	time (s)
1	98	10
10	98	1
	62 ↓ 0.5	5
	72	120
20	98	1
	57	5
	72	120
1	72	300
1	12	-
NI15/16 in T100
cycles	temperature (°C)	time (s)
1	98	10
30	98	1
	65	5
	72	120
1	72	300
1	12	-
KH5/6 in C1000
cycles	temperature (°C)	time (s)
1	98	10
10	98	1
	63 ↓ 0.5	5
	72	10
30	98	1
	59	5
	72	10
1	72	30
1	12	-
NI17/18 in C1000
cycles	temperature (°C)	time (s)
1	98	10
10	98	1
	63 ↓ 0.5	5
	72	10
30	98	1
	65	5
	72	10
1	72	30
1	12	-
KH7/8 and NI19/20 in BioRad old one
cycles	temperature (°C)	time (s)
1	98	10
30	98	1
	65	5
	72	10
1	72	30
1	12	-

Agarose Gel and Gel Extraction

For NI15/16 wrong template (pMM64) was used in the first PCR. The second run (pKH1/2) yielded the right product fragments have been gel extracted using QIAquick Gel extraction kit. NI15/16 and KH3/4 have been extracted using

QiaEXII gel extraction kit.

CPEC assembly and Transformation

CPEC assembly of CCDB fragment (KH1/2 and NI13/14) and indigoidine synthesis construct (pRB3-delta-T, pKH1-delta-T

and pKH2-delta-T to yield pRB11, pRB12 and pRB13. As a positive control, native T-Domains (KH5/6 and NI19/20) are

reinserted. Transformation in ccdb resistant strain (OneShot CCDB survival cells) using standard protocol and 5 ul

of CPEC reaction product.

CPEC assembly Phusion Flash HF in biometra cycler
cycles	temp (°C)	time (s)
1	98	10
10	98	1
	55	5
	72	120
1	72	300
1	12	-

On plates with colonies are always blue colonies as well as white ones -> no information on assembly success.

CCDB screening and test

Five white colonies from pRB12 were picked as well as a blue colony as a control. Conlony PCR using intron iTaq

polymerase was performed and 6x 0.5 mL LB+Cm were inoculated with the six colonies.

ccdb screening pRB12 colonies with NI13/14 in
cycles	temperature (°C)	time (s)
1	98	10
35	98	60
	60	30
	72	80
1	72	300
1	12	-

Screening is positive -> miniprep of colonies #3 and #4 from 2 ml liquid culture. Purified plasmid pRB12 was transformed to TOP10, DH5alpha and OneShot on LB+Cm to check CCDB functionality. OneShot should grow whereas plates of DH5alpha and TOP10 were expected to stay empty since ccdb causes cell death.

All strains grow on LB+Cm which shows that ccdb doesn't work as expected. ccdb with RBS was amplified from pDONR

plasmid.

Results and Discussion

Assembly and/or Transformation were inefficient, blue colonies on each plate suggest that a lot of template was in

the Transformation mix. The positive control doesn't provide information on whether the assembly was successful.

CCDB screenig of pRB12 was positive but transformation in DH5alpha, TOP10 and OneShot ccdb survival cells showed

that ccdb is unfunctional. In the following, all the insert from pDONR will be used including a promoter and the

ccda gene. We will work with indC on pSB1C3 with PPTases on separate plasmids to be able to easily test different

combinations of T-Domain and PPTase.

Lab book

===CPEC Conditions Assay (Ralf)=== We performed CPEC assembly of pRB13 with eight different CPEC conditions to see whether we get difeerent results.

Table 11.1 CPEC mix for assembly of pRB13

Annealing Temp [°C]	Cycles
	5	10	15
53		2: 10 ul; 4 ul backbone; 1 ul CCDB
55	3: 10 ul; 4 ul backbone; 1 ul CCDB 7: 20 ul; 8.5 ul backbone; 1.5 ul CCDB	4: 10 ul; 4 ul backbone; 1 ul CCDB 6: 20 ul; 8.5 ul backbone; 1.5 ul CCDB 8: 10 ul; 4.5 ul backbone; 0.5 ul CCDB	5: 10 ul; 4 ul backbone; 1 ul CCDB
57		1: 10 ul; 4 ul backbone; 1 ul CCDB

Table 11.2 CPEC Assembly of pRB13

BioRad T100
cycles	temp [°C]	time [s]
1	98	10
5/10/15	98	1
	53/55/57	5
	72	120
1	72	300
1	12	-

100 ul of competent TOP10 were transformed with 5 ul of reaction product and incubated for 30 hours at 37 °C.

Apparently there is no big difference using various CPEC conditions. We will continue with the standard protocol.

PPTase test (Ralf)

pRB3-10 are plasmids containing indC (pRB-3-6) or bpsA(pMM64) (pRB7-10) and a PPTase (sfp, svp, svp(pMM65), entD).

This week the blue colonies are screened and sequenced for validation.

colony PCR screening #1

Two blue colonies are picked from each plate and screened with the primers of the PPTase they should have and the

ones which they could have if something went wrong. PPTase PCR products werde used as a positive control and TOP10

without any plasmid as a negative control. So numbers 5 to 18 are subdivided into alpha and beta.

	PPTase to be screened
template	sfp (RB35/36)	svp (RB29/30)	svp(pMM65) (RB25/26)	entD (RB33/34)
	1
		2
			3
				4
	5
		6
			7
	13	14	15	8
	9
		10
			11
	16	17	18	12
	19	20	21	22

BioRad T100
cycles	temp [°C]	time [s]
1	120
30	95	60
	65	30
	72	60
1	72	300
1	12	-

Loading Scheme
5a	5b	9a	9b	1	19	2-log	6a	6b	10a	10b	2	20
7a	7b	11a	11b	3	21	2-log	8a	8b	12a	12b	4	22

The gel is not conclusive. Therefore I will repeat the screening but screen every colony for all PPTases.

MiniPrep of pRB4-10

5 ml liquid cultures of TOP10+pRB4-10 have been miniprepped using QIAquick miniprep kit. Only blue cultures have

been prepped (except pRB9 because there was no blue culture). pRB4-6 and pRB10 were prepped in duplicates (liquid

cultures of two different clones) whereas there are only single preps of pRB7-9. This is because pRB3-6 contain

indc ahich we will use for further studies. pRB6 and 10 contain entD, which is the E. coli endogenous PPTase and

was previously declared to be "inefficient" in activating bpsA [Takahashi 2007].

Concentrations of the prepped plasmids have been measured using ??ThermoScientific NanoDrop

plasmid	version	concentration [ng/ ul]
pRB4	alpha	289.7
pRB4	beta	278.7
pRB5	alpha	228.3
pRB5	beta	348.9
pRB6	alpha	428.0
pRB6	beta	432.2
pRB7	alpha	187.3
pRB8	alpha	768.2
pRB9	alpha	142.5
pRB10	alpha	112.0
pRB10	beta	212.0
water after measurement		0.7

Minipreps were reduced to a concentration of about 50 ng/ ul for sequencing and 1 ng/ ul for further studies.

MiniPrep PCR screening #2

	PPTase to be screened
template	sfp (RB35/36)	svp (RB29/30)	svp(pMM65) (RB25/26)	entD (RB33/34)
TOP10+pRB4	5	6	7	8
TOP10+pRB5	9	10	11	12
TOP10+pRB6	13	14	15	16
TOP10+pRB7	17	18	19	20
TOP10+pRB8	21	22	23	24
TOP10+pRB9	25	26	27	28
TOP10+pRB10	29	30	31	32
sfp	33
svp		34
svp(pMM65)			35
entD				36
TOP10	37	38	39	40

BioRad T100
cycles	temp [°C]	time [s]
1	95	120
30	95	60
	60	30
	72	60
1	72	300
1	12	-

Loading Scheme
5a	5b	9a	9b	13a	13b	17	21	25	29a	29b	sfp	2-log	6a	6b	10a	10b	14a	14b	18	22	26	30a	30b	svp
7a	7b	11a	11b	15a	15b	19	23	27	31a	31b	svp(pMM65)	2-log	8a	8b	12a	12b	16a	16b	20	24	28	32a	32b	entD

pRB9 is negative. All screens are positive in the svp(pMM65) and entD section. In entD this could be due to genomic

DNA in the miniprep. svp(pMM65) is also positive in all plasmids, which is strange. Maybe the PCR conditions have

been too tolerant. Nevertheless the bands of the plasmids which should carry the respective PPTase appear to be

stronger. We will sequence the plasmids anyways.

Sequencing of pRB3-10

plasmid minipreps have been sequenced by GATC [reference] using the sequencing primers VF2 and VR. The sequencing

results show that all the inserts are correct except for pRB9, which isn't a pity because we have previously shown

with pKH1-2 that the combination of bpsA(pMM64) and svp(pMM65) successful produces indigoidine.

plasmid	Primer	ind synthetase/ PPTase	sequencing result/ reference
pRB3	VF2	indC
	VF2	indC	seq2
	VR	sfp-rev
	VR	sfp-rev	seq2
pRB4	VF2	indC
	VF2	indC	seq2
	VR	svp(pMM65)-rev
	VR	svp(pMM65)-rev	seq2
pRB5	VF2	indC
	VF2	indC	seq2
	VR	svp-rev
	VR	svp-rev	seq2
pRB6	VF2	indC
	VF2	indC	seq2
	VR	entD-rev
	VR	entD-rev	seq2
pRB7	VF2	bpsA
	VF2	bpsA	seq2
	VR	sfp-rev
	VR	sfp-rev	seq2
pRB8	VF2	bpsA
	VF2	bpsA	seq2
	VR	svp(pMM65)-rev
	VR	svp(pMM65)-rev	seq2
pRB9	VF2	bpsA
	VF2	bpsA	seq2
	VR	svp-rev
	VR	svp-rev	seq2
pRB10	VF2	bpsA
	VF2	bpsA	seq2
	VR	entD-rev
	VR	entD-rev	seq2

In the following pRB4-beta, pRB5-alpha, pRB5-beta, pRB6-alpha and pRB10-alpha have been selected so that there are

single versions of pRB3-10 for furhter experiments.

Glycerols stocks pRB3-10

3 stocks each were made using 2 mL liquid cultures (48 h at 37 °C 200 rpm) of TOP10+pRB3-10, respectively.

Lab book

Preparation for T-Domain exchanges

We assemble pRB14, which is a version of pRB3, in which the T-Domain is exchanged by the whole insert of the pDONR vector. Since pRB13 grew on DH5alpha and TOP10 we hope that pRB14 won't. This vector will be used to exchange T-Domains and will be modified to get a version without the PPTase sfp and the cutting sites EcoRI and SpeI in indC. To check whether a successfully exchanged T-Domain can be activated by a defined PPTase, we build pSB2K3-derived plasmids with a single PPTase (sfp, svp, entD or delC) under control of the lac-Promotor and the RBS BBa_B0029.

Fragment Amplification

PCR of Fragments for PPTase plasmids

Table 12.x PCR for pRB15-18: 25 ul Phusion Flash HF MM 2x; 5 ul Primer 10 uM each; template according to table; water ad 50 ul.

pSB2K3 RB21/63 in BioRAD old old
template: 3 ul pSB2K3 250 ng/ ul (distribution)
cycles	temperature (°C)	time (s)
1	98	10
10	98	1
	62 ? 0.5	5
	72	40
25	98	1
	57	5
	72	40
1	72	300
1	12	-

pSB2K3 contains the 3'-part of the reversed Primer in an internal lac-Operator-structure, so the PCR product is just half of the backbone (2500 bp instead of 4800 bp). We will use the same primers but pSB3K3 of the 2013 spring distribution plate 5 well 5E; which is around 3000 bp.

Table 12.x PCR for pRB15-18: 25 ul Phusion Flash HF MM 2x; 5 ul Primer 10 uM each; template according to table; water ad 50 ul.

pSB3K3 RB21/63 in BioRAD old old
template: 2 ul pSB3K3 250 ng/ ul (distribution)
cycles	temperature (°C)	time (s)
1	98	10
3	98	1
	58	5
	72	50
32	98	1
	65	5
	72	50
1	72	180
1	12	-

We get unspecific product -> repetition with more stringent conditions

pSB3K3 RB21/63 in BioRAD T100
template: 0.5/1.5 ul pSB3K3 250 ng/ ul (distribution)
cycles	temperature (°C)	time (s)
1	98	10
10	98	1
	65 -0.5	5
	72	50
25	98	1
	69	5
	72	50
1	72	180
1	12	-

PCR of Fragments for T-Domain exchange

Table 12.x PCR for pRB13: 25 ul Phusion Flash HF MM 2x; 5 ul Primer 10 uM each; template according to table; water ad 50 ul.

pRB3?T KH3/4 in T100
template: 1 ul pRB3 1 ng/ ul
cycles	temperature (°C)	time (s)
1	98	10
10	98	1
	62 ? 0.5	5
	72	120
25	98	1
	57	5
	72	120
1	72	300
1	12	-
KH9/10 in T100
template: 2 ul pDONR 1 ng/ ul
cycles	temperature (°C)	time (s)
1	98	10
8	98	1
	70 ? 0.5	5
	72	15
28	98	1
	65	5
	72	15
1	72	90
1	12	-

T-Domains in BioRAD old
tycC6-T RB57/58 1 ul B. para overnight culture
tycA1-T RB55/56 1 ul B. para overnight culture
entF-T RB53/54 colony MG1655
delH4-T RB59/60 colony D. aci SPH-1
delH5-T RB61/62 colony D. aci SPH-1
cycles	temperature (°C)	time (s)
1	98	120
40	98	1
	65	5
	72	5
1	72	30
1	12	-

Gel Extraction

Gel extraction was performed using QIAquick gel extraction kit. DNA yield was measured using NanoDrop Spectrophotometer ND-1000

Table 12.x DNA concentrations for assembly of pRB-T

Fragment	Concentration [ng/ ul]	~ Fragment size [bp]	Molarity [nM]
sfp	329.6	700	713.42
svp	44.9	750	90.7
entD	289.2	650	674.13
delC	54.5	700	117.97
pSB3K3	26.8	3000	13.54
pRB3dT	124.8	6800	27.81
ccdB	121.2	700	262.33
indC-T	192.4	200	1457.58
bpsA-T	198.9	200	1506.18
entF-T	184.8	200	1400.00
tycA1-T	184.2	200	1395.45
tycC6-T	198.6	200	1504.55
delH4-T	175.0	200	1325.76
delH5-T	162.4	200	1230.30

CPEC Assembly and Transformation

pRB14-18

Plasmid	Fragment 1	Molarity [nM]	Volume in MM	Fragment 2	Molarity [nM]	Volume in MM
pRB14	pRB3dT	27.81	3.8	ccdB	262.33	1.2
pRB15	pSB3K3	13.54	4.5	sfp	713.42	0.5
pRB16	pSB3K3	13.54	3.5	svp	90.7	1.5
pRB17	pSB3K3	13.54	4.5	entD	674.13	0.5
pRB18	pSB3K3	13.54	3.8	delC	117.97	1.2
pRB21	indC(RB27/46)	25.40	3.5	pSB1C3(RB21/22)	133.08	1.5

Table 12.x

CPEC Assembly pRB14-18

Cycles	Temperature [°C]	Time [s]
BioRAD T100
1	98	10
5	98	1
	53	5
	72	120
1	72	300
1	12	-

Transforation according to standard protocol for chemical transformation

TOP10 with pRB15-18 (-> LB+Kan)
TOP10 with pRB15-18 and pMM64, respectively (-> LB+Kan+Amp)
TOP10 with pMM64 (-> LB+Amp)
OneShot with pRB14 (-> LB+Cm)

OneShot+pRB14: small colonies after 20 hours
TOP10+pRB15-18: colonies on all plates
TOP10+pMM64+pRB15-18: (almost) no colonies after 20 hours; hopefully due to indigoidine growth retardation
TOP10+pMM64 TraFo was efficient

We will perform PCR screening in triplicates of TOP10+pRB15-19 and OneShot+pRB14 to get a first impression on whether the assembly was successful.

Plasmid Validation

PCR Screening

We use forward primers of the insert and standard reversed primer VR to screen pRB14-18 in triplicates. We use iTaq DNA-polymerase in 20 ul PCR mix:

10 ul iTaq 2x Master Mix
2 ul Primer 10 uM each
6 ul water
colony pick from plate

Plasmid	Primer fw	Primer rv	Expected Fragment Size [bp]
pRB14	VF2	KH10	1000
pRB15	VF2	RB36	1050
pRB16	VF2	RB30	1100
pRB17	VF2	RB34	1000
pRB18	VF2	RB67	1050

Table 12.x

PCR screening pRB14-18

Cycles	Temperature [°C]	Time [s]
BioRAD T100
1	95	120
35	95	30
	53	30
	72	60
1	72	300
1	12	-

Annealing temperature according to NEB Tm calculator for Taq DNA polymerase and VF2

Heidelberg 20130814 pRB14-18 screen2.PNG

Table 12.x concentrations of pRB15-18 (NanoDrop ND-1000)
Plasmid	Concentration [ng/ ul]	~ size [bp]
pRB15	98.7	3500
pRB16	106.6	3500
pRB17	188.3	3500
pRB18	129.9	3500

Test Transformation

OneShot and TOP10-sells have been transformed with pRB14. We expect OneShot cells to grow as usual and no colonies on TOP10. As a control group we transformed OneShot as well as TOP10 with pRB3.

pRB19

pRB19 is a pRB14-derived plasmid without the PPTase sfp. The genotype is pSB1C3-lacPromotor-BBa_B0034-indCdT(ccdB)

Table 12.x

PCR Amplification of pRB14 for pRB19

BioRAD T100
RB21/46 from pRB14 miniprep 0.6 ng
Cycles	Temperature [°C]	Time [s]
1	98	10
8	98	1
	TD 61	5
	72	100
30	98	1
	65	5
	72	100
1	72	300
1	12	-

Smear -> Annealing temperatures in the touchdown-part were too high, so RB21 3' end couldn't bind properly. Second run with milder conditions.

Table 12.x

PCR Amplification of pRB14 for pRB19 #2

BioRAD T100
RB21/46 from pRB14 miniprep 1 ng
Cycles	Temperature [°C]	Time [s]
1	98	10
12	98	1
	TD 60	5
	72	110
25	98	1
	65	5
	72	110
1	72	300
1	12	-

Smear -> we will run another strategy, i.e. amplification of indC from pRB14 with RB27/46 to further assemble it with pSB1C3(RB21/22)

Table 12.x

PCR Amplification of indC(RB27/46) for pRB19 #3

BioRAD T100
RB27/46 from pRB14 miniprep 1 ng
Cycles	Temperature [°C]	Time [s]
1	98	10
10	98	1
	TD 57	5
	72	60
25	98	1
	65	5
	72	60
1	72	180
1	12	-

Heidelberg 20130816 indC(RB27-46) pRB14 3.PNG

Heidelberg 20130816 indC(RB27-46) pRB14 3 cut.PNG

Gel extraction was performed using QIAquick gel extraction kit. DNA yield was measured using NanoDrop Spectrophotometer ND-1000

Table 12.x DNA concentrations for assembly of pRB19

Fragment	Concentration [ng/ ul]	~ Fragment size [bp]	Molarity [nM]
indC-pRB14(RB27/46)	133.0	3800	53.03
pSB1C3(RB21/22)	210.8	2400	133.08

Plasmid	Fragment 1	Molarity [nM]	Volume in MM
Plasmid	!Fragment 2	Molarity [nM]	Volume in MM
pRB19	indC-pRB14(RB27/46)	pRB14(RB21/46)	53.03	3.3
pRB19	pSB1C3(RB21/22)	133.08	1.7

Table 12.x

CPEC Assembly pRB19

Cycles	Temperature [°C]	Time [s]
BioRAD T100
1	98	10
5	98	1
	53	5
	72	100
1	72	300
1	12	-

Transformation into OneShot Survival cells. Doing colony PCR for screening with primer KH9/VR which would give a fragment size of around 1.8 kbp: (Note: Throw picked colony 4 away since unintendently pooled 2 colonies in PCR tube). Used iTaq.

Table 12.x

PCR screening pRB19 and pRB21

Cycles	Temperature [°C]	Time [s]
Cycler 2
KH9/VR from picked colonies.
1	95	120
35	95	30
	53	30
	72	120
1	72	300
1	12	-

colony PCR for pRB19 and pRB21

Colony PCR for pRB19 gave a lot of unspecific bands, probably because of less stringent parameters. Oculutated 6 ml of TB+Cm with colony 2 for MP preparation.

Biobricks

The PPTases sfp, svp, entD and delC and the indigoidine synthetase indC will be prepared for submission to the registry. indC contains two RFC10-cutting site, that have been removed during the assembly of pRB22.

pRB21 series

pRB21 is a pSB1C3-plasmid with lacPromotor, BBa_B0034 and indC. After the first assembly, EcoRI and SpeI cutting sites in indC will be removed to yield pRB22. After cutting site removal, the native T-Domain will be exchanged with ccdB to yield pRB22. pRB23 has the same genotype as pRB20 but with two PCR steps less.

Fragment Amplification

Table 12.x

PCR Amplification of indC for pRB21 #1

BioRAD T100
RB27/46 from P. luminescens pellet
Cycles	Temperature [°C]	Time [s]
1	98	10
8	98	1
	TD 61	5
	72	120
25	98	1
	65	5
	72	120
1	72	300
1	12	-

Amplification was not successful. Next try from PCR product with short primers as a rePCR.

Table 12.x

PCR Amplification of indC for pRB21 #2

BioRAD T100
RB27/46 from indC short gel extraction
Cycles	Temperature [°C]	Time [s]
1	98	10
8	98	1
	TD 56	5
	72	60
25	98	1
	65	5
	72	60
1	72	180
1	12	-

Heidelberg 20130815 PPTBBa indCpRB21.PNG

Heidelberg 20130815 PPTBBa indCpRB21 cut2.PNG

Gel Extraction

Gel extraction was performed using QIAquick gel extraction kit. DNA yield was measured using NanoDrop Spectrophotometer ND-1000

Table 12.x DNA concentrations for assembly of pRB21

Fragment	Concentration [ng/ ul]	~ Fragment size [bp]	Molarity [nM]
indC(RB27/46)	63.7	3800	25.40
pSB1C3(RB21/22)	210.8	2400	133.08

CPEC Assembly and Transformation

Plasmid	Fragment 1	Molarity [nM]	Volume in MM	Fragment 2	Molarity [nM]	Volume in MM
pRB21	indC(RB27/46)	25.40	3.5	pSB1C3(RB21/22)	133.08	1.5

Table 12.x

CPEC Assembly pRB21

Cycles	Temperature [°C]	Time [s]
BioRAD T100
1	98	10
5	98	1
	53	5
	72	60
1	72	180
1	12	-

Validation

Colony PCR for pRB21 was unsuccessful (see gel picture), try again with less strigent parameters and positive control: KH5/VR on MP pRB3, which should give an amplicon size of around 2 kbp.

Table 12.x

PCR screening pRB21

Cycles	Temperature [°C]	Time [s]
T100 (right)
KH5/VR from picked colonies and 5 ng pRB3 MP
1	95	120
35	95	30
	50	30
	72	90
1	72	150
1	12	-

colony PCR forpRB21

Colony PCR for pRB21 worked now as well as the positive control. Oculutated 6 ml of TB+Cm with colony 8 for MP preparation.

PPTase BioBricks

have to look up used primer/template combinations again Konrad (talk)

Table 12.x

PCR Amplification of PPTases for BioBrick submission

BioRAD T100
sfp: KH17/18 from pRB15
svp: KH19/20 from pRB16
entD: KH13/14 from pRB17
delC: KH15/16 from pRB18
Cycles	Temperature [°C]	Time [s]
1	98	10
12	98	1
	TD 66	5
	72	15
25	98	1
	65	5
	72	15
1	72	60
1	12	-

Heidelberg 20130815 PPTBBa indCpRB21 cut2.PNG

Gel Extraction

Assembly and Transformation

Digest

Ligation

=====Transformation=====

Lab book

Preparation for T-Domain exchange

Fragment Amplification

Table 12.x PCR for T/TE-Domain exchange with CPEC. Phusion Flash HF
cycles	temperature (°C)	time (s)
template: 0.5 ul from pRB19 miniprep, Primer KH3/4
template: 0.5 ul from pRB19 miniprep, Primer KHRB21/KH4
1	98	10
35	98	1
	60	5
	72	100
1	72	300
1	12	-

PCR #1 for fragment for T-Domain exchange

No band at 6400 bp. Second try with less stringent conditions

Table 12.x PCR #2 KH3/4 from pRB19, Phusion Flash HF
cycles	temperature (°C)	time (s)
template: 0.5 ul from pRB19 miniprep, Primer KH3/4
template: 0.5 ul from pRB19 miniprep, Primer KHRB21/KH4
1	98	10
10	98	1
	62 ? 0.5	5
	72	100
25	98	1
	57	5
	72	100
1	72	300
1	12	-

PCR #2 for fragment for T-Domain exchange

Heidelberg 20130820 pRB19 1 TTE2 cut.png

Still nothing; maybe the MiniPrep didn't work so fine. We will screen pRB19-transformed cells on pRB19. We want to get a first impression on whether the Domain-exchange works, so we'll use pRB14 instead.

Table 12.x PCR #2 KH3/4 from pRB14, Phusion Flash HF
cycles	temperature (°C)	time (s)
template: 0.8 ug from pRB14 miniprep, Primer KH3/4
template: 0.8 ug from pRB14 miniprep, Primer KHRB21/KH4
1	98	10
10	98	1
	62 ? 0.5	5
	72	120
25	98	1
	57	5
	72	120
1	72	300
1	12	-

PCR #3 for fragment for T-Domain exchange

Heidelberg 20130820 pRB14 entF-TTE cut.png

PCR worked but with very low yield. We try to amplify from pRB19-transformed cells instead of the miniprep.

Table 12.x PCR #2 KH3/4 from pRB19, Phusion Flash HF
cycles	temperature (°C)	time (s)
template: 1 ul from pRB19-transformed cells, Primer KH3/4
template: 1 ul from pRB19-transformed cells, Primer KHRB21/KH4
1	98	10
30	98	1
	57-60-68	5
	72	120
1	72	300
1	12	-

25 ul Phusion HF MM 2x; 5 ul Primer 10 uM each; template according to table; water ad 50 ul

Table 12.x Table 12.x PCR TTE-domains
TTE-Domains in BioRAD cycler 2
bpsA-TTE KH7/RB72 1 ng pMM64 miniprep
entF-TTE RB53/73 colony MG1655
tycC6-TTE RB57/74 1 ul B. para overnight culture
delH5-TTE RB61/75 colony D. aci SPH-1
cycles	temperature (°C)	time (s)
1	98	120
40	98	10
	65	20
	72	30
1	72	120
1	12	-

PCR #1 for TTE-Domains

bpsA-TTE and tycC6-TTE worked, the other ones will be amplified using less stringent conditions

25 ul Phusion Flash HF MM 2x; 5 ul Primer 10 uM each; template according to table; water ad 50 ul

Table 12.x Table 12.x PCR TTE-domains
TTE-Domains in BioRAD T100
entF-TTE RB53/73 colony MG1655
delH5-TTE RB61/75 colony D. aci SPH-1
cycles	temperature (°C)	time (s)
1	98	120
10	98	1
	62 ? 0.5	5
	72	20
25	98	10
	60	5
	72	20
1	72	60
1	12	-

PCR #2 for TTE-Domains

delH5-TTE worked, though with low yield. entF-TTE will be amplified in a third PCR.

25 ul Phusion Flash HF MM 2x; 5 ul Primer 10 uM each; template according to table; water ad 50 ul

Table 12.x Table 12.x PCR TTE-domains
TTE-Domains in BioRAD T100
entF-TTE RB53/73 colony MG1655
cycles	temperature (°C)	time (s)
1	98	120
10	98	1
	60 ? 0.5	5
	72	20
25	98	10
	55	5
	72	20
1	72	60
1	12	-

PCR #3 for TTE-Domains

entF-TTE didn't work. We will postpone this PCR.

T-Domain exchange

CPEC Assembly and Transformation

Plasmid	Fragment 1	Molarity [nM]	Volume in ul	Fragment 2	Molarity [nM]	Volume in MM	CPEC Master Mix total [ul]
pRB14-T1	pRB14d(ccdB)	3.1	2.7	indC-T	1457.6	0.3	6
pRB14-T2	pRB14d(ccdB)	3.1	2.7	bpsA-T	1506.1	0.3	6
pRB14-T4	pRB14d(ccdB)	3.1	2.7	tycA1-T	1395.5	0.3	6
pRB14-T5	pRB14d(ccdB)	3.1	2.7	tycC6-T	1504.5	0.3	6
pRB14-T6	pRB14d(ccdB)	3.1	2.7	delH4-T	1325.8	0.3	6
pRB14-T7	pRB14d(ccdB)	3.1	2.7	delH5-T	1230.3	0.3	6
pRB14-TTE1	pRB14d(ccdB-TE)	33.5	3	bpsA-TTE	140.8	2	10
pRB14-TTE3	pRB14d(ccdB-TE)	33.5	1.5	tycC6-TTE	31.8	3.5	10
pRB14-TTE4	pRB14d(ccdB-TE)	33.5	1.5	delH5-TTE	34.7	3.5	10

Table 13.x CPEC Assembly pRB14-Tx/TTEx
Cycles	Temperature [°C]	Time [s]
BioRAD T100
1	98	10
8	98	1
	53	5
	72	120
1	72	300
1	12	-

Transformation in TOP10 according to standard protocol. Incubation for 30 hours at 37 °C and 10 hours

on the bench.

BioBricks

Fragment Preparation

25 ul Phusion Flash HF MM 2x; 5 ul Primer 10 uM each; template according to table; water ad 50 ul

Table 12.x PCR for pRB22
cycles	temperature (°C)	time (s)
miniprep of pRB22 with RB68/69
1	98	10
35	98	1
	60	5
	72	100
1	72	300
1	12	-

PCR #1 for fragment for T-Domain exchange

PCR worked well. To make sure that we don't transform template plasmid pRB21, we digest the pRB22d(EcoRI-SpeI) fragment Gel Extraction together with pRB22d(SpeI-EcoRI) with DpnI for four hours at 37 °C.

Table 13.x DpnI-digest for pRB22
Component	Concentration	Volume [ul]
37 °C; 4 hours
pRB22d(EcoRI-SpeI)	32.3 nM	11.0
pRB22d(SpeI-EcoRi)	839.4 nM	2.4
CutSmart buffer	10x	1.6
NEB DpnI		1

The Digest was purified using QiaGen Nucleotide Removal Kit. Unfortunately I dropped 2/3 onto the table... The final DNA concentration was 47.5 ng/ ul according to NanoVue. Standard CPEC assembly was performed in the old broken BioRAD cycler which didn't cool down to 53 °C but only to 58 °C for the annealing step. Hopefully it worked. TOP10 cells were transformed with 5 ul of CPEC master mix.

Transformation was successful. Ten colonies were screened using primers VF2 and KH6 in a 20 ul iTaq Master Mix according to standard protocol (50 °C).

Probes number x to x were purified using Qiaquick PCR purification kit and digested with NEB DpnI, EcoRI-HF and SpeI-HF over night to see whether pRB22 is really free of those restriction sites. The result was put on an AgaroseGel.

Lanes 1 to 4 show a slight smear beneath 200 bp. Lane 5 (probe 10) doesn't show that smear and the

specific band runs maybe 300 bp higher. So maybe the first four lanes contained restriction sites and

the last one didn't. We put probes 1 and 10 in 40 ml culture for midiprep. The midiprep will then be

digested as well so that we get certainity about the restriction sites. In parallel we did a colony PCR from probe 10 with KH3/4 using Phusion Flash HF to already have our

fragment for pRB23 for the T-Domain-exchange.

Table 13.x PCR for pRB23
cycles	temperature (°C)	time (s)
miniprep of pRB23 with RB68/69
1	98	120
10	98	1
	TD 62	5
	72	140
25	98	1
	57	5
	72	120
1	72	300
1	12	-

Lab book

Preparation for T-Domain exchange

Assembly of pRB19

PCR indC(ccdB) RB27/28 and pSB1C3 RB21/22 25 ul Phusion Flash HF MM 2x; 5 ul Primer 10 uM each; template according to table; water ad 50 ul

Table 14.x PCR indC(ccdB) for pRB19
RB27/46 in BioRAD T100
cycles	temperature [°C]	time [s]
1	98	10
5	98	1
	55	5
	72	60
30	98	1
30	72	60
1	72	180
1	12	-

Table 14.x PCR indC(ccdB) for pRB19
RB27/46 in BioRAD T100
cycles	temperature [°C]	time [s]
1	98	10
10	98	1
	td 55	5
	72	70
25	98	1
	60	5
	72	70
1	72	180
1	12	-

Gel Extraction with QIAquick gel extraction kit.

Table 13.x CPEC Assembly Master Mix pRB19 second try
Plasmid	Fragment 1	Molarity [nM]	Volume in ul	Fragment 2	Molarity [nM]	Volume in MM	DpnI Master Mix total [ul]
pRB19	indC(ccdB) from pRB14	14.6	14	pSB1C3 RB21/22	105.3	3	20

Digest ran for 1 hour at 37 °C, was put on a agarose gel, gel extracted and eluted in 15 ul water.

Concentration was measured using NanoVue: 30 ng/ ul. 10 ul were put together with 10 ul of Phusion

Flash HF Master Mix for standard CPEC assembly. OneShot and TOP10 cellc were transformed using different amounts of CPEC product.

Ten colonies were screened using iTaq with KH9/VR and RB35/VR, respectively, to see whether the assembly worked and sfp was kicked out.

The screening was positive, so probe 10 was kept for 10 ml liquid culture (TB+Cm). Miniprep of 4 ml yielded 69.4 ng/ ul in 50 ul. Miniprep was used for PCR with primers KH3/4 to get the fragment pRB19 without ccdB for insertion of T-Domains via CPEC. First we tried two different polymerases at two different conditions each. The first is Phusion Flash

HF 2x Master Mix (ThermoScientific) and the other is Phusion HF 2x Master Mix (NEB).

Table 14.x Test PCR KH3/4 from pRB19
KH3/4 in BioRAD T100 with Phusion HF (NEB)
Cycles	Temperature [°C]	Time [s]
1	98	30
30	98	15
	57-60	15
	72	3.30
1	72	10.00
1	12	-
KH3/4 in BioRAD T100 with Phusion Flash HF (ThermoScientific)
Cycles	Temperature [°C]	Time [s]
1	98	10
30	98	1
	57-60	5
	72	2.00
1	72	6.00
1	12	-

Table 14.x synT-PCR
KH5/6 in BioRAD T100
cycles	temperature [°C]	time [s]
1	98	30
45	98	5
	60	15
	72	15
1	72	60
1	12	-

Gel extraction and measurement using NanvoVue. The final table of T-domain concentration is as

follows:

Table 13.x Concentrations of T- and TTE-domains to be shuffled
Fragment	Concentration [ng/ ul]	~ Fragment size [bp]	Molarity [nM]
ccdB	121.2	700	262.33
indC-T	192.4	200	1457.58
bpsA-T	198.9	200	1506.18
entF-T	64.5	200	488.6
tycA1-T	184.2	200	1395.45
tycC6-T	198.6	200	1504.55
delH4-T	175.0	200	1325.76
delH5-T	162.4	200	1230.30
plu2642-T	79.0	200	598.5
plu2670-T	31.5	200	238.6
synT1	43	200	325.8
synT2	37.5	200	284.1
synT3	40.5	200	306.8
synT4	36	200	272.7
synT5	37.5	200	284.1
synT6	37	200	280.3
synT7	40.5	200	306.8
bpsA-TTE	92.9	1000	140.8
entF-TTE	-	1000	-
tycC6-TTE	21	1000	31.8
delH5-T	22.9	1000	34.7

Assembly of pRB15-18

pRB15-18 were unable to be sequenced with various primers. We now use pSB3K3 backbone from plate 3 of the 2013 spring distribution and assemble those PPTase-plasmids de novo.

Table 14.x PCR pSB3K3 with RB21/63 #1
RB21/63 in BioRAD T100
Cycles	Temperature [°C]	Time [s]
1	98	10
14	98	1
	td 66	5
	72	60
16	98	1
	68	5
	72	60
1	72	180
1	12	-

PCR was run with with a fragment of the Tyrocidine group with slightly suboptimal conditions. It will be repeated using otimal conditions.

Table 14.x PCR pSB3K3 with RB21/63 #2
RB21/63 in BioRAD T100
Cycles	Temperature [°C]	Time [s]
1	98	10
12	98	1
	td 61	5
	72	50
20	98	1
	65	5
	72	50
1	72	180
1	12	-

Gel extraction yielded 144.2 ng/ ul (Nanodrop)

CPEC was performed using pSB3K3 gel extraction and gel extractions made for assembly of pRB3-10. TOP10 cells were transformed using standard protocol.

Table 14.x CPEC Assembly pRB15-18
Plasmid	Fragment 1	Molarity [nM]	Volume [ul]	Fragment 2	Molarity [nM]	Volume [ul]	CPEC Master Mix total [ul]
pRB15	pSB3K3(RB21/63)	87.4	2	sfp	713.4	1	6
pRB16	pSB3K3(RB21/63)	87.4	2	svp	90.7	4	12
pRB17	pSB3K3(RB21/63)	87.4	2	entD	674.1	1	6
pRB18	pSB3K3(RB21/63)	87.4	1	delC	118.1	2	6

Cells were plated on LB+Kanamycin and incubated at 37 °C for 24 hours.

Five colonies of each plate were screened using iTaq polymerase and two primer combinations for each colony. This is VF2/PPTase_rv primer and PPTase_fw/VR primer.

pRB16 is obviously wrong. We checked the PCR product we used for the assembly and recognized that it's wrong, so we have to amplify it again from S. verticillus. DelC screenings show another small band, we have too check whether the primers bind elsewhere on the plasmid and run a negative control with those primers.

Assembly of pKH4

pKH4 is a pRB3 derived version of an indigoidine synthetase expression plasmid w/o sfp as activating PPtases. So the two plasmid strategy can be conducted (at first) with this plasmid instead to rely on pRB21. Basically the idea is to use restriction sites before and after sfp to discard it and to religate with another sequence to yield a functional backbone.

pRB3 has a BamHI before and a NheI cutting site after sfp. pMM64 and pMM65 both can be cut with a combination of BglII and XbaI to yield for example a 164 bp long fragment with compatible ends to the linearized pRB3. This fragment unfortunately contains a T7lac hybrid promoter which could interfere with our used primers, but still is worth a shot.

digest mix volume: 30 µl (3 µl NEBuffer 3.1, 6.5 µl pMM65 (~481 ng), 0.3 µl (BglII, XbaI) each, 19.9 µl H2O)

Resulting gel of digestion of pMM65 with BglII and XbaI in order to gain sequence with cohesive ends to linerized pRB3 w/o sfp.

digestion gave expected bands at 2 kbp and 2.4 kbp but band at ~160 bp is missing. Reaction was carried out with not enough DNA.
prepared ON for pMM64/65 MP as well as pRB3 MP
the next day ON were extended to 2x 4 ml LB cultures which were then

prepped after several hours of incubation at 37 °C

MP with cultures: DNA conc. measurement with NanoVue gave: 280

ng/µl (pMM64), 225 ng/µl (pMM65), 250 ng/µl (pRB3); analysis gel shows different concentration for pMM65 (~15 ng/µl ?)

Analytical gel for verification of MP pMM64/65 and pRB3: concentrations measured with NanoVue seem to be ok for pMM64 and pRB3, but much less for pMM65. 2log ladder lane contain 420 ng each; 100 V, TAE

prepare digestion (20 µl), 37 °C, 1.5 h:
- pMM64: 2 µl NEBuffer 3.1, 17 µl pMM64, 0.5 µl (BglII, XbaI)
- pRB3: 2 µl NEBuffer CutSmart, 10 µl pRB3, 0.5 µl (BamHI, NheI), 7

µl H2O

Digestion of pMM64 (BglII, XbaI) and pRB3 (BamHI, NheI) were desired bands for insert of ~170 bp and backbone-indC of ~6300 bp were cut out. 100 V, TAE

after gelextraction (elution in 20 µl) ligation was conducted with 2

µl 10x T4 ligase buffer, 1 µl T4 ligase, 2 µl pRB3 Dig_GE, 8 µl pMM64 Dig_GE and 7 µl H2O for 40 min at RT

Analytical gel of 1 µl of ligation mix, compared to 0.5 µl of pRB3 digestion with (BamHI, NheI). Additionally 1 µl of MP pMM65 was put on gel to verify previous concentration measurement. 1.5 µl of log2 ladder, 100 V, TAE

transformation in TOP10 with 1 µl and 5 µl of ligation mix

Lab book

pRB15-18

The first mini

Screening delC

Table 15.x Screening pRB18
Template	Primer	Comment
TOP10+pSB3K3	RB64/VR	negative control
TOP10+pSB3K3	VF2/65	negative control
TOP10+pRB18	RB64/VR	screening
	VF2/65
	VF2/VR
delC PCR product	RB64/RB65	positive control

Screening pKH4

Table 15.x Screening pKH4
Template	Primer
TOP10+pKH4	KH52/VR
TOP10+pKH4	VF2/RB71

A miniprep of probe 4 was sent to sequencing, which was positive.

Assembly of pKH4-derivates

pKH4 is used for assembly of pKH5 and variants of pKH4 with all the different T-Domains.

Table 15.x Amplification KH3/4 from pKH4
KH3/4 in BioRAD T100
cycles	temperature [°C]	time [s]
1	98	10
10	98	1
	td 62	5
	72	2.00
25	98	1
	57	5
	72	2.00
1	72	6.00
1	12	-

KH3/4-PCRs were unsuccessful, so we did a gradient PCR

Table 15.x Amplification KH3/4 from pKH4
KH3/4 in biometra
cycles	temperature [°C]	time [s]
1	98	10
10	98	1
	58-59-60-61-62-63	5
	72	2.00
1	72	6.00
1	12	-

We run the same protocol for 3x 50 ul.

nothing

16 x 10 ul with Q5 and Tm calculator suggested parameters (63 °C annealing), pRB19 ligation as template

We tried NEB Phusion HF with 61 °C annealing temperature in a 10-, 20- and 50 ul reaction from pRB19 ligation and in a 20 ul reaction from pKH4 ind T100 (left). As well we ran with Phusion HF (59 °C annealing) in 20 ul reactions with 0, 1 and 2 ul DMSO (Biorad Cycler 2)

For further evaluation of the single plasmid strategy we admired a plasmid with bpsA instead of indC as indigoidine synthetase. For a quick shot we wanted to use pKH4 and pRB7 for a restriction approach with the enzymes KpnI and BamHI.

prepare digestion (20 µl), 37 °C, ~0.5 h:
- pKH4: 2 µl NEBuffer CutSmart, 4 µl pKH4, 13 µl H2O, 0.5 µl (KpnI, BamHI)
- pRB7: 2 µl NEBuffer CutSmart, 2.3 µl pRB7, 14.7 µl H2O, 0.5 µl (KpnI, BamHI)

Digestion of pKH4 (KpnI, BamHI) and pRB7 (KpnI, BamHI). Desired bands for insert of ~3.8 kbp and backbone+sfp of ~3.9 kbp were expected. 100 V, 0.8 % Agarose, TAE

The restriction for pKH4 did not work correctly due to the fact that with assembly of pKH4 the BamHI cutting site was removed and the plasmid was only linearized by the KpnI cutting site. For pRB7 two bands at almost equal site can be expected and thereby the digestion approach can not be used in this manner. We will try again with a similar approach like for the assembly of pKH4 as here a nonsense sequence instead of the PPtases will be introduced into pRB7.

preparation of pRB7 MP by retransformation of TOP10 (as no glycerol stock is available).

Amplification of svp

The svp fragment we used so far was wrong, so we try to amplify it again from the genome, using various primers:

RB13/14
RB15/16
RB29/30
RB39/40

Gradient on biometra cycler (72 °C, 65 °C, 58 °C)

svp was always right; only after assembly it turned wrong. We will try to bring it in using restriction cloning and to sequence it; maybe the screening is just inconclusive.

Amplification of indC-fragments

indC fragments are amplified from Plum genome and indC short PCR product in a 50 ul Phusioin HF reaction. The fragments are:

RB27/69
RB70/71
RB68/46
RB68/KH4
KH5/RB46
RB27/46 from pRB14
RB27/28

Table 14.x PCR indC(ccdB) for pRB19
RB27/69 in BioRAD T100 from indC short
RB27/69 in BioRAD T100 from P. luminescens pellet
RB70/71 in BioRAD T100 from indC short
RB70/71 in BioRAD T100 from P. luminescens pellet
KH5/RB46 in BioRAD T100 from indC short
KH5/RB46 in BioRAD T100 from P. luminescens pellet
cycles	temperature [°C]	time [s]
1	98	2.00
35	98	5
	55	15
	72	30
1	72	2.00
1	12	-
RB68/46 in BioRAD T100 from indC short
RB68/46 in BioRAD T100 from P. luminescens pellet
RB68/KH4 in BioRAD T100 from indC short
RB68/KH4 in BioRAD T100 from P. luminescens pellet
cycles	temperature [°C]	time [s]
1	98	2.00
30	98	15
	55	15
	72	2.00
1	72	5.00
1	12	-

Heidelberg 20130902 indC-fragments cut.png

Fragment RB70/71 is wrong because RB71 is wrong. We repeated the amplification with RB78 instead of RB71.

Fragment KH3/RB46 is very weak, so we try to improve this reaction as well.

Table 15.x Amplification indC RB21/46 from pRB14 and pSB1C3 RB21/22 from miniprep
KH3/RB46 in BioRAD T100
cycles	temperature [°C]	time [s]
1	98	10
12	98	1
	td 60	5
	72	20
23	98	1
	63	5
	72	20
1	72	60
1	12	-

And reaction RB68/RB46 with the same protocol but 60 s elongation time.

We try KH3/RB79 with improved conditions.

Heidelberg 20130903 RB79-fragments cut.png

We tried to get this fragment with KH9/RB79 with Q5 in biometra cycler (60 °C annealing)

We tried to get this fragment with KH9/RB79 with Phusion HF in biometra old cycler (61 °C annealing)

Gel Extraction yielded xx ng/ ul

CPEC assemblies were performed for following plasmids with the respective fragments:

pRB19: RB27/79 from pRB14 and RB21/22 from pSB1C3 midiprep
pRB22: RB27/69 RB70/78 RB68/79 RB21/22
pRB23: RB27/69 RB70/78 RB68/KH4 KH9/RB79 (pRB14) RB21/22
pRB24: RB27/28 RB66/67 RB21/22
pKH5: KH3/4 (pKH4) KH9/10

Transformed cells looked as follows:

Colony screenings

pRB23 assembly was unsuccessful, pRB22 is correct (plates turn blue), pRB24 seems to be correct (small deep blue colonies), pRB19 didn't work, pKH5 seems correct. We prepped the marked colonies. Sequencing showed that pRB22 is correct and doesn't carry endogenous RFC10 cutting sites any longer. Test digestion with EcoRI and SpeI also enforced this result.

We performed a PCR with Phusion HF and KH3/4 from pRB22, assembled it in a CPEC approach with ccdB KH9/10 and transformed OneShot cells.

Colony screenings and test-transformation in TOP10 cells showed that the assembly was successful and that we're able to shuffle the T-domains.

Minipreps

Minipreps of pRB15, pRB17, pRB18, pKH4 and pRB14 yielded:

pRB15
pRB17
pRB18
pRB14: 284.8 ng/ ul in 20 ul
pKH4: 316.8 ng/ ul in 20 ul

5 ul of each pRB15, 17 and 18 were put on an agarose gel with small pockets.

PPTase plasmid minipreps are empty. We will try to assemble them again using a restriction strategy besides prepping them again. Repetitioin of the miniprep was successful and the plasmids were sent to sequencing.

Cotransformation

We did cotransformation of pKH4 with pRB15-18, respectively, and single transformation of pSB3K3, pKH4 and pRB15-18.

Restriction cloning for pRB15-19

Table 15.x Amplification indC RB21/46 from pRB14 and pSB1C3 RB21/22 from miniprep
RB21/22 in BioRAD T100
cycles	temperature [°C]	time [s]
1	98	10
12	98	1
	td 60	5
	72	50
23	98	1
	65	5
	72	40
1	72	3.00
1	12	-
RB27/46 in BioRAD T100
cycles	temperature [°C]	time [s]
1	98	10
12	98	1
	td 60	5
	72	70
18	98	1
	65	5
	72	60
1	72	2.00
1	12	-

svp was reamplified from short product

Table 15.x Amplification indC RB21/46 from pRB14 and pSB1C3 RB21/22 from miniprep
RB21/22 in BioRAD T100
cycles	temperature [°C]	time [s]
1	98	10
35	98	1
	65	5
	72	20
1	72	60
1	12	-

indC and pSB1C3 were digested with KpnI and NheI, sfp, svp, entD, delC and pSB3K3 with BamHI and NheI for two hours at 37 °C.

Unfortunately pSB3K3 contains an internal NheI cutting site. We repeated the amplification, purification, restriction digest and purification with BamHI and SpeI, which builds a scar with NheI.

Ligation of pSB1C3 with indC(ccdB) was performed over night in a 10 ul reaction with 4 ul fragments each.

Transformation of ligation mixes were performed with 5 µl each in TOP10. Additionally 5 µl of ligation mix of pRB19 was transformed into OneShot cells.

Colony screenings showed following results:

We prepped pRB15, 17 and 18 and repeated the assembly of pRB16 from scratch. Therefore we used RB13/14 for amplification of svp from an S. verticilllus ATCC15003 liquid culture. Afterwards we reamplified this PCR gel extraction with RB29/30 and performed a restriction digest with BamHI and NheI to ligate the fragment with pSB3K3 (BamHI; SpeI).

Reevaluation of old MP pRB15-18, pRB21

Because we could observe small colonies with darkgray center on cotransformed pRB21 with all PPtases constructs (plates of 2013-08-27), we conducted a colony screen and a retransformation with 1 µl of previous MP of pRB15.2 (Konrad) and pRB16-18 (Ralf) and pRB21 (Ralf). Additionally a cotransformation with this old pRB21 MP with the old pRB15.2 as well with 2.5 µl of newly assembled pRB15 was performed.

The colony screening was performed using the primers VR and KH5. If the gray colonies carry two plasmids with indigoidine synthetase and activating PPtase separately an amplicon of size ~1.3 kbp. Otherwise if for some reason the PPtase and indigoidine synthetase are still on one plasmid, we could expect an amplicon of 2.1 kbp.

Table 15.x colony screening cotrafo pRB21 and pRB15-18, 2013-08-27
KH5/VR in Biometra, 10 µl
cycles	temperature [°C]	time [s]
1	95	120
30	95	30
	60	30
	72	120
1	72	240
1	12	-

Colony PCR of cotransformation pRB21 and pRB15-18. Desired bands would be expected at 1.3 kbp for positive clones. For positive controls amplicons were expected at ~2 kbp for pRB3 and at ~1.5 kbp for pKH4. 100 V, 0.8 % Agarose, TAE

The positive controls gave the expected bands. All picked clones also gave an expected band at 1.3 kbp which leads to the conclusion that they carry the indC-only plasmid pRB21. Because every third clone was a picked white colony we could also conclude, that not all transformants carry an intact PPtase plasmid.

T-domain exchange with different domain borders

We thought of a different way to set the T-Domain borders. Starting from the Pfam-predicted domain borders we made pairwise alignments to find amino acid sequences up- and downstream the predicted borders, that are identical in both the native and the brought in sequence. In the case of the exchange of indC and bpsA those new borders would be around 10 amino acids more on each side. We designed primers for this strategy.

Screenings

Table 15.x Screening of new constructs
Plasmid	Primer 1	Primer 2	Expected product [bp]	wrong product [bp]	additional test
pKH6	VR	RB17	1070
pKH7	VR	RB13	1120
pKH8	VR	RB41	1000
pKH9	VR	RB64	1070
pRB15_lig	VR	RB17	1070
pRB16_lig	VR	RB13	1120
pRB17_lig	VR	RB41	1000
pRB18_lig	VR	RB64	1070
pRB19_lig	VR	KH9	1900	2600
pRB19_lig	VR	RB17	-	1070
pRB22_frag					digest
pRB23_frag	VR	KH9	1900		digest
pRB23_frag	RB27	KH4	2900
pKH5	VR	KH9	2100
pRB24	VR	KH5	2600
pRB24	VR	RB64	1070
pRB7	-	-	-	-	digest

We repeated the screening of pKH8 with 20 colonies and amplified svp with RB29/30 from pKH7.

Lab book

===pRB23-T variants=== We amplified pRB23ΔccdB with KH3/4 in 2x 50 ul Phusion HF reactions and did a gel extraction.

We performed CPEC assembly using 2.7 ul of the KH3/4 fragment and 0.3 ul of each of the T-Domain PCR products, which have been amplified some weeks ago. E. coli TOP10 were transformed with 5 ul of the CPEC reaction mix. The transformants were incubated at 37 °C over night. We picked colonies to inoculate liquid cultures for the preparation of plasmid DNA. The prepped plasmid were then used for co-transformed with each of the PPTase plasmids pRB15-18 as well as without a PPTase. The following table shows the transformants after four days at room temperature in the drawer.
synT1, synT3 and synT4 result in blue colonies!!! We sequenced the prepped plasmids using the VR. They are all correct.

pRB15-19

Transformation of TOP10 cells with the new assembled pRB16.3 variant.

Colony screening of six clones with VF2 and the svp reverse primer showed following result:

We prepped colony "3" and performed the first bulk of cotransformations with it. Sequencing of the PPTase plasmids we used for the cotransformations were positive except that we found a point mutation in pRB16.3 which results in a stop codon right in the middle of svp. Therefore we will prepare the five other colonies except number 2 and screen 21 more colonies.

We prepped colonies 19 and 26 and cotransformed TOP10 cells together with pRB22. The shape of the colonies will show us whether the PPTase is correct or carries the stop codon.

Quantitative Analysis of Indigoidine production - Assay developpement

Find best ODs to measure

We perform spectral analysis of an E. coli TOP10+pRB22+pRB15 over night liquid culture in an Amersham Biosciences Ultrospec 3300 pro. Pure TOP10 liquid culture is used as a reference and pure LB medium for blanking. The absorption spectrum hsows a significant maximum at about 590 nm.

Upscaling

To be able to increase throughput we will use a Tecan infinite M200 plate reader for our growth kinetics and indigoidine production measurement. In a first test run we measure the OD at the wavelengths 400 nm, 530 nm, 590 nm and 800 nm every 5 minutes. The liquid cultures in this case are:

TOP10 negative control
TOP10 + pRB22
TOP10 + pRB3
MG1655 + pRB3

By this we get an impression how we can detect and quantify both the total indigoidine production per well and the indigoidine production in relation to the cell density. The measurement properties can be seen in the following figure.

Lab book

Quantitative Assay

Measurement

This week we go for a first big measurement with our engineered indigoidine synthetases. The following figure is a scheme of our 96 well plate for the first measurement. We used one plate to grow positive (i.e. indigoidine producing) E. coli TOP10 transformants from an agar plate in liquid culture for 30 hours at 37 °C. We prepared a second 96 well plate with 180 ul media and the respective antibiotics an inoculated with 20 ul of the liquid culture from the first plate.
The second plate was observed in our Tecan plate reader. This time we measured the absorbance from 400 nm to 800 nm in 2 nm steps and 25 reads for every well. This configuration unfortunately leads to the problem that the reader takes a long time for each measurement and we get huge time intervals between each measurement. Therefore we stopped the measurement after aboout 12 hours and changed the configuration. For the next 15 hours we used an interval of 10 nm and used 25 reads per well, so we are able to get a higher resolution on the time scale.

The following table shows the loading scheme of our 96 well plate in this run.

96-well	1	2	3	4	5	6	7	8	9	10	11	12
A	-	-	-	-	-	-	-	-	-	-	-	-
B	-	-	-	-	-	Mücke/ Spinne	-	-	MG1655+pRB3	TOP10+pRB3	BAP1+pRB3	-
C	-	TOP10	-	pRB22	pRB23-T2	pRB23-T8	pRB23-T10	pRB23-T12	pRB23-T13	pRB23-T14	pRB23-T16	-
D	-	TOP10+pRB15	pRB3	pRB22+pRB15	pRB23-T2+pRB15	pRB23-T8+pRB15	pRB23-T10+pRB15	pRB23-T12+pRB15	pRB23-T13+pRB15	pRB23-T14+pRB15	pRB23-T16+pRB15	-
E	-	TOP10+pRB16	pRB4	pRB22+pRB16	pRB23-T2+pRB16	pRB23-T8+pRB16	pRB23-T10+pRB16	pRB23-T12+pRB16	pRB23-T13+pRB16	pRB23-T14+pRB16	pRB23-T16+pRB16	-
F	-	TOP10+pRB17	pRB6	pRB22+pRB17	pRB23-T2+pRB17	pRB23-T8+pRB17	pRB23-T10+pRB17	pRB23-T12+pRB17	pRB23-T13+pRB17	pRB23-T14+pRB17	pRB23-T16+pRB17	-
G	-	TOP10+pRB18	pRB24	pRB22+pRB18	pRB23-T2+pRB18	pRB23-T8+pRB18	pRB23-T10+pRB18	pRB23-T12+pRB18	pRB23-T13+pRB18	pRB23-T14+pRB18	pRB23-T16+pRB18	-
H	-	-	-	-	-	-	-	-	-	-	-	-

Results

We created plots showing the OD absorption versus wavelength for all timepoint from 0 to 30 hours (top). The plot down left is the OD590 versus OD800. This plot should be linear for every negative liquid culture which doesn't produce indigoidine. The last plot shows the OD of the indigoidine without the cells versus the OD800 from the liquid culture, thus showing how much indigoidine is produced ind a liquid culture at a specific OD.

Lab book

BioBrick Characterization

Test Digest

We digested 400 ng of each of our parts [http://parts.igem.org/Part:BBa_K1152008 BBa_K1152008] to BBa_K1152019 with EcoRI, XbaI, SpeI and PstI. We used 0.5 ul enzyme and incubated the reaction at 37 °C for 2 hours. We submitted the plasmids pKH6-10, pRB22, pRB23, pRB23-T8, -T10, -T12, -T13 and -T14.

The digest is positive and the fragment sizes are correct. Together with the sequencing results we can be sure that our submitted parts are alright.

Lab book

Domain Shuffling - Indigoidine Conversions

This week we assemble 28 plasmids pRB23-ds1112 to pRB23-ds3132, which are variants of indC, in which single domains are exchanged by the corresponding domains of plu2642, a single module NRPS of unknown function from P. luminescens and tycC2 from the tyrocidine cluster. The A-domains of both modules were predicted to be specific for glutamine binding and activation using the NRPS Designer. We will assemble all the domain combinations listed below. The plasmid code consissts of pRB23-ds and a code with four numbers. Each number defines the origin of the domain at the respective positions: indC is "1", plu2642 is "2" and tycC2 is "3". ds1122 for example means that in the general sequence of A-OxA-T-TE the A and OxA domain come from indC, whereas the T and TE domain are the ones of plu2642.

The following table shows the fragments the pRB23-dsxxxx plasmids consist of:

Table 19.1: Possible domain combinations using domains of indC (number1 in the *dsxxxx* code), plu2642 (2) and tycC2 (3)
	Fragment 1	Primer_fw	Primer_rv	Fragment 2	Primer_fw	Primer_rv	Fragment 3	Primer_fw	Primer_rv	Fragment 4	Primer_fw	Primer_rv	Fragment 5	Primer_fw	Primer_rv
pRB23-ds1112	indC BB GE	RB27	KH6	Plum	RB110	RB100	pRB23	RB21	RB22
pRB23-ds1121	pRB23	KH3	KH4	Plum	RB97	KH12
pRB23-ds1122	pRB23	RB21	KH4	Plum	RB97	RB100
pRB23-ds1131	pRB23	KH3	KH4	Bpara	RB105	RB106
pRB23-ds1132	pRB23	RB21	KH4	Bpara	RB105	RB106	Plum	RB110	RB100
pRB23-ds2022	pRB23	RB21	RB22	Plum	RB99	RB100
pRB23-ds2111	Plum	RB99	RB101	indC BB GE	RB102	RB79	pRB23	RB21	RB22
pRB23-ds2112	Plum	RB99	RB101	indC BB GE	RB102	KH6	Plum	RB110	RB100	pRB23	RB21	RB22
pRB23-ds2121	Plum	RB99	RB101	pRB23	RB102	KH4	Plum	RB97	KH12	pRB23	KH3	RB22
pRB23-ds2122	Plum	RB99	RB101	pRB23	RB102	KH4	Plum	RB97	RB100	pRB23	RB21	RB22
pRB23-ds2131	Plum	RB99	RB101	pRB23	RB102	KH4	Bpara	RB105	RB106	pRB23	KH3	RB22
pRB23-ds2132	Plum	RB99	RB101	pRB23	RB102	KH4	Bpara	RB105	RB106	Plum	RB110	RB100	pRB23	RB21	RB22
pRB23-ds3111	Bpara	RB107	RB108	indC BB GE	RB102	RB79	pRB23	RB21	RB22
pRB23-ds3112	Bpara	RB107	RB108	indC BB GE	RB102	KH6	Plum	RB110	RB100	pRB23	RB21	RB22
pRB23-ds3121	Bpara	RB107	RB108	pRB23	RB102	KH4	Plum	RB97	KH12	pRB23	KH3	RB22
pRB23-ds3122	Bpara	RB107	RB108	pRB23	RB102	KH4	Plum	RB97	RB100	pRB23	RB21	RB22
pRB23-ds3131	Bpara	RB107	RB108	pRB23	RB102	KH4	Bpara	RB105	RB106	pRB23	KH3	RB22
pRB23-ds3132	Bpara	RB107	RB108	pRB23	RB102	KH4	Bpara	RB105	RB106	Plum	RB110	RB100	pRB23	RB21	RB22

All fragments are amplified with Phusion HF (NEB) in 40 ul reactions in three thermocyclers and purified using agarose gel extraction.

Table 19.2: PCR fragments for assembly of pRB23-dsxxxx.
Fragment	Template	Primer_fw	Primer_rv	Usage	Fragment size [bp]	Annealing [°C]	Elongation [s]	x 40 ul MM
C_1	pRB23	RB21	RB22	8	2500	65	70	8
C_2	pRB23	KH3	RB22	4	3300	65	70	4
C_3	pRB23	RB102	RB22	2	5000	65	120	2
C_4	pRB23	RB21	RB81	2	2900	65	70	2
C_5	pRB23	KH3	RB81	2	6000	65	120	2
C_6	pRB23	RB102	RB811	8	1400	65	30	8
C_7	indC PCR product from pRB22	RB27	KH6	1	3100	65	70	1
C_8	indC PCR product from pRB22	RB102	KH6	2	1700	65	30	2
P_1	P. luminescens cell pellet	RB99	RB101	6	1500	65	30	6
P_2	P. luminescens cell pellet	RB97	KH12	3	300	65	30	2
P_3	P. luminescens cell pellet	RB110	RB100	6	850	65	30	4
P_4	P. luminescens cell pellet	RB97	RB100	3	1150	65	30	3
P_5	P. luminescens cell pellet	RB99	RB100	1	2800	65	70	1
T_1	B. parabrevis cell pellet	RB107	RB108	6	1450	65	30	6
T_2	B. parabrevis cell pellet	RB105	RB106	6	300	65	30	3

The fragments then will be used for the CPEC assembly of all plasmids. CPEC is performed in 20 ul reactions for each plasmid so that it is sufficient for three transformations and an analytical agarose gel electrophoresis.

Table 19.3: CPEC Assembly of pRB23-dsxxxx ind a 20 ul reaction
CPEC	Fragment 1	~ conc. [nM]	Size [bp]	Volume in CPEC [ul]	Fragment 2	~ conc. [nM]	Size [bp]	Volume in CPEC [ul]	Fragment 3	~ conc. [nM]	Size [bp]	Volume in CPEC [ul]	Fragment 4	~ conc. [nM]	Size [bp]	Volume in CPEC [ul]	Fragment 5	~ conc. [nM]	Size [bp]	Volume in CPEC [ul]
pRB23-ds1112	C_7	9,78	3100	7,0	P_2	404,04	300	1,0	C_1	90,91	2500	2,0	-	-	-	-	-	-	-	-
pRB23-ds1121	C_5	0,51	6000	9,0	P_2	404,04	300	1,0	-	-	-	-	-	-	-	-	-	-	-	-
pRB23-ds1122	C_4	0,00	2900		P_4	39,53	1150		-	-	-	-	-	-	-	-	-	-	-	-
pRB23-ds1131	C_5	0,51	6000	9,0	T_2	757,58	300	1,0	-	-	-	-	-	-	-	-	-	-	-	-
pRB23-ds1132	C_4	0,00	2900		T_2	757,58	300		P_3	35,65	850		-	-	-	-	-	-	-	-
pRB23-ds2022	P_5	2,16	2800	8,5	C_1	90,91	2500	1,5	-	-	-	-	-	-	-	-	-	-	-	-
pRB23-ds2111	P_1	4,04	1500	5,5	C_8	53,48	1700	1,5	C_2	18,37	3300	3,0	-	-	-	-	-	-	-	-
pRB23-ds2112	P_1	4,04	1500	5,0	C_8	53,48	1700	2,0	P_3	35,65	850	2,0	C_1	90,91	2500	1,0	-	-	-	-
pRB23-ds2121	P_1	4,04	1500	6,0	C_6	162,34	1400	1,0	P_2	404,04	300	1,0	C_2	18,37	3300	2,0	-	-	-	-
pRB23-ds2122	P_1	4,04	1500	6,0	C_6	162,34	1400	1,0	P_3	35,65	850	2,0	C_1	90,91	2500	1,0	-	-	-	-
pRB23-ds2131	P_1	4,04	1500	6,0	C_6	162,34	1400	1,0	T_2	757,58	300	1,0	C_2	18,37	3300	2,0	-	-	-	-
pRB23-ds2132	P_1	4,04	1500	6,0	C_6	162,34	1400	1,0	T_2	757,58	300	1,0	P_3	35,65	850	2,0	C_1	90,91	2500
pRB23-ds3111	T_1	20,90	1450	4,0	C_8	53,48	1700	2,0	C_2	18,37	3300	3,0	-	-	-	-	-	-	-	-
pRB23-ds3112	T_1	20,90	1450	4,0	C_8	53,48	1700	2,0	P_3	35,65	850	3,0	C_1	90,91	2500		-	-	-	-
pRB23-ds3121	T_1	20,90	1450	4,0	C_6	162,34	1400	1,0	P_2	404,04	300	1,0	C_2	18,37	3300	4,0	-	-	-	-
pRB23-ds3122	T_1	20,90	1450	4,0	C_6	162,34	1400	1,0	P_3	35,65	850	3,5	C_1	90,91	2500	1,5	-	-	-	-
pRB23-ds3131	T_1	20,90	1450	4,0	C_6	162,34	1400	1,0	T_2	757,58	300	1,0	C_2	18,37	3300	4,0	-	-	-	-
pRB23-ds3132	T_1	20,90	1450	4,0	C_6	162,34	1400	1,0	T_2	757,58	300	1,0	P_3	35,65	850	4,0	C_1	90,91	2500

We transform NEB Turbo cells with each plasmid (5 ul of the CPEC reaction product) and cotransform the cells with each plasmid and pRB15 and pRB17, respectively.

Thanks to

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Team:Heidelberg/Indigoidine

From 2013.igem.org

Revision as of 16:34, 25 October 2013

Team

Project

Notebook

Parts

Software

Human Practice

Safety

Modeling

Indigoidine. Proving Modularity of NRPS by Shuffling Domains.

Week 5

Indigoidine Production - bpsA

Week 6

Indigoidine Production - bpsA

Week 7

Indigoidine Production - bpsA

Week 8

Indigoidine Production - bpsA

Week 9

Indigioidine Production – bpsA

Week 10

Indigoidine Production - bpsA

Week 11

Indigoidine Production - bpsA

Week 12

Indigoidine Production - indC

Week 13

Indigoidine Production – Testing PPTases

Week 14

T-Domain Shuffling – Assembly of ccdB Plasmids

Week 15

Indigoidine Production – Testing PPTases

Week 16

T-Domain Shuffling – PPTase Plasmids

BioBricks for the Registry

Week 17

T-Domain Shuffling

T-Domain Shuffling – PPTase Plasmids

BioBricks for the Registry

Week 18

T-Domain Shuffling

BioBricks for the Registry

Week 19

Domain Shuffling – PPTase Plasmids

T-Domain Shuffling

Week 20

T-Domain Shuffling

T-Domain Borders

T-Domain Shuffling – PPTase Plasmids

Engineered indC - Quantitative Assay

Week 21

BioBricks for the Registry

Engineered indC – Quantitative Assay

Week 22

T-domain shuffling

Controlling Indigoidine synthesis

Week 23

Domain Shuffling – Indigoidine Synthetase Conversions

Engineered indC – Quantitative Assay

Indigoidine production with pMM-plasmids I (Ilia)

Results and Discussion

Indigoidine production with pMM-plasmids II (Ilia)

Results and Discussion

Indigoidine production with pMM-plasmids III (Ilia)

Indigoidine production with pMM-plasmids IV (Ilia)

Fragment amplification for pKH1 (Konrad)

Assembly of pKH1 and Transformation (Konrad)

Gibson approach

CPEC approach

Transformation

Colony PCR

analytical digestion of selected clones

validation of transformed BAP1 with Fussenegger plasmids

fusion PCR

Gibson Assembly of bpsA + svp insert

PCR for colony validation

IPTG induction, titration

IPTG induction, titration RESULT