Team:Aachen/Notebook
Lab Book
To express a photocaged protease in our case subtilisin E, we developed three lab strategies. Their milestones can be seen below in three different timelines, one for every sub-division. Details about reaching these milestones will become visible, if you click on the corresponding circles. On the page "Project Idea" the theory beneath our lab work is described and on the subpage "Protocols & Methods" the exact protocols can be seen, if you would like to repeat the experiments.
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Expression of subtilisin E
As the first step to reach our aim of producing an inactive photocaged Subtilisin E variant, which can be activated by light, we researched on the recombinant expression of Subtilisin E in S. cerevisiae . The native Subtilisin E is a gene of Bacillus subtilis which we could receive from the institute. In the following steps our procedures can be traced.
Determination of Parameters
Strain selection
S. cervisiae
Synthetase is made for this strand
needs to have the deficiencies of both chosen vectors
Characterization of strand [1]
| Name | Auxotrophies | Prototrophies | Mating Type |
| CENPK2-1D | his3D1; leu2-3_112; ura3-52; trp1-289 | MAL2-8c; SUC2 | MATalpha |
Growth – temperature
We tested the growth in different temperatures for optimization:
| Temperature | OD at start | OD after 24 h |
| 25°C | 0 | 0 |
| 30°C | 0 | >2 |
| 37°C | 0 | 0 |
The S. Cerevisiae strand only grew at 30°C.
Growth – media
As our CENPK2-1D strand was growing poorly in the SC-U medium, we got the advice to try the same medium but with a higher amount of amino acids (SD-U).
| Medium | OD at start | OD after 24 h |
| SC-U | 0,063 | 2,914 |
| SC-U | 0,072 | 9,14 |
After OD measurements it was obvious, that the SD-U medium gave the CENPK2-1D better conditions for growth
Growth – protection against contamination
To prevent bacterial contaminations, we made a comparison of the growth of our used strand in medium with and without ampicillin.
| Medium | OD at start | OD after 24 h |
| YPD | 0 | 2,725 |
| YPD+Amp | 0 | 2,61 |
The growth in both media seems equal. So we decided to only use medium with additional ampicillin in future.
Selection of vector
Requirements for vector
1. Secretion
Subtilisin E must be secreted as we do not know it it can be expressed active in cytoplasm for industry secretion is a big advantage as it allow continuos productoin
2. Shuttle vector
For amplifying the vector easily it needs to have an ori and selective marker in E. coli
3. Selection System in yeast different than Synthetase Plasmid
Auxotrphy must be different from trytophane
Available vector for us:
| Vector | pTEF-MF | pYES2 | pCFP255 [1] | pYES2Mfalpha | pCFP255MfalphaTEF1 | pESC-Trp |
| selection in S. cerevisiae | ura3 | ura3 | ura3 | ura3 | ura3 | trp |
| selection in E. coli | Amp | Amp | Amp | Amp | Amp | Amp |
| Secretiont | yes | no | no | yes | yes | no |
| Genome Integration | no | no | yes | no | yes | no |
| Length | 6,6 kb | 5,9 kb | - | 6,2 kb | - | 6,5 kb |
| Promoter | TEF1 | GAL | TEF1 | GAL | TEF1 | GAL |
References
[1]http://www.euroscarf.de/plasmid_details.php?accno=30000B
[2]Jensen et al. (2013). “EasyClone: method for iterative chromosomal integration of multiple genes in Saccharomyces cerevisiae” FEMS Yeast Research 14(2): 238-248. Provided by Irina Borodina, Technical University of Denmark
Cloning
Cloning of Subtilisin E Gene from Bacillus subtilis into an E. coli - S. cerevisiae shuttle vector
In the beginning we received the PHY300PKL plasmid with the Subtilisin E gene. We chose an uracil deficient Saccharomyces cerevisiae strand and a shuttle vector carrying the URA gene. The vector has the secretion tag MF alpha to make harvesting of produced protein more convenient. After cloning the Subtilisin E gene into the vector in Escherichia coli we want to transform it in S. cerevisiae and make a test expression. A scheme of the cloning can be seen below:
For the detailed labbook entries see this PDF.
Cloning of Codon Optimized Subtilisin E in pTEF-MF
As the test expression of the Subtilisin E gene from bacillus subtilis was not successful we used a codon optimized variant of the gene we had on the PHY300PKL plasmid. As a host we chose the S. Cerevisiae strand CENPK2-1D again as it has the deficiencies we need for selection. As a vector backbone we tried the pTEF-MF vector again. A scheme of the cloning can be seen below:
For the detailed labbook entries see this PDF.
Cloning into the inducible vector pYes2
After day 10 of cloning the codon optimized Subtilisin E into pTEF-MF we tried a parallel strategy in the PYES2 vector. A scheme of the cloning can be seen below:
For the detailed labbook entries see this PDF.
Genome Integration (pCFB255) [1]
After codon optimisation we planned to have the subtilisin E inside an integrative vector pCFB255 for better expression in Saccharomyces cerevisiae
For the detailed labbook entries see this PDF.
Test Expression
Introduction
After cloning the Subtilisin E into the vectors pTEF-MF, pyes2 and the genome integration vector pCfB255 a test expression was made. We used the following methods to test for proteolytic activity:
Skim Milk assay
The skim milk assay is a non specific test for proteolytic activity. Lysis of casein leads to a clear solution while the solution remains white when the proteolytic activity is missing.
AAPF assay
The AAPF assay is a very specific test for proteolytic activity in serine proteases
TCA precipitation
It is used to precipitate proteins from cell culture supernatant to gain a higher concentration.
SDS gel
An SDS gel can be used to make proteins visible. SDS (Sodiumdodecyl-sulfate) charges all proteins negative, so that they migrate in an electric field. It is a size based sorting method.
Results in pTEF-MF
In the pTEF-MF the secretion tag MFalpha should lead to a secretion.
We tested the supernatant of 24h, 48h and 72h cultures with the methods described above but did not get any positive results.
We also crushed the cells to test them with the same methods after 24h, 48h and 72h but there was no positive result either.
Subtilisin E expression was not possible in this expression system.
Results in pyes2
In the pyes2 vector with additional MFalpha secretion tag Subtilsin E also should be secreted.
We tested the vector like pTEF-MF except we induced it first with 20 % Galactose and fed 20 % again after every 24h.
Results in pcfB255
Tests were performed just like the pTEF-MF, but also genome integration did not result in secretion of an active protease.
SDM Ser to UAG
Quikchange of Subtilisin E Gene
Introduction
To make insertion of the DMNB-serine into the enzyme possible, an amber stop codon has to be at the respective site. In order to achieve this, we made a quikchange of our gene.
All protocols we used can be seen under this link
For all agarose gels we used 1% agarose and 1 kb Thermo scientific ladder 08:
Day 1
SDM Subtilsin E: Serine 221 to amber codon
We made a SDM with YP0010 and YP0011. As template we used 1µl of sequenced Subtilisin E in pTEF-MF (day 14 from cloning of Subtilisin E gene from Bacillus subtilis) with a concentration of 13,6 ng/µl per 50 µl reaction.
Programm 1 (cycles: 3)
| ID | 98°C | 30 sec |
| D | 98°C | 10 sec |
| A | 65-74°C | 45 sec |
| E | 72°C | 6 min |
| S | 8°C | storage |
Programm 2 (cylces: 15)
| ID | 98°C | 30 sec |
| D | 98°C | 10 sec |
| A | 65-74°C | 45 sec |
| E | 72°C | 5 min |
| FE | 72°C | 10 min |
| S | 8°C | storage |
We used colums 4-9
| Colum | 4 | 5 | 6 | 7 | 8 | 9 |
| Temperature | 66,4°C | 67,4°C | 68,6°C | 69,8°C | 71°C | 72,1°C |
Agarose Gel
We made an agarose gel to see if our modified Subtilsin E was amplified with the primers. The expected size is 7700bp.
1: PCR product of column 4
2: PCR-product of column 5
3: PCR-product of column 6
4: PCR-product of column 7
5: PCR-product of column 8
6: PCR-product of column 9
0: ladder
There are clear bands at all the six annealing temperatures, so the PCR worked well.
DPN1 Digestion
We did a DPN1 digestion overnight of the quik change products.
Day 2
PCR-Clean-Up
We made a PCR-clean-up of the DPN1 digested products to prepare them for transformation in E. coli Dh5alpha.
Transformation in DH5alpha
We transformed the purified SDM product into E. coli DH5 alpha and plated them on LB + Amp plates.
Day 3
Transformation Results
The transformation was successful.
Overnights
We made overnights of 4 of the grown colonies in 5 ml LB + Ampicillin.
Day 4
Plasmid Isolation
We isolated the plasmids of the quikchanged Subtilisin E in pTEF-MF overnights.
Nanodrop
We measured the concentration of isolated plasmids via nanodrop:
1. 912,6 ng/µl
2. 910 ng/µl
3. 866 ng/µl
4. 660,4 ng/µl
Sequencing
We sent the four samples of isolated plasmids in for sequencing with YP0007, YP0008 and YP0009.
Day 5
Sequencing Results
All four samples showed the right sequence.
Results
We did not continue after this step as the Subtilisin E expression did not work.
Test Expression
As our previous efforts did not produce usable results, we did not reach this point.
Expression of Synthetase
Determination of Parameters
Check genes
Test Expression
Co-transformation
As our previous efforts did not produce usable results, we did not reach this point.
Test Expression
As our previous efforts did not produce usable results, we did not reach this point.
Test Photoclevage and Activity
As our previous efforts did not produce usable results, we did not reach this point.
New BioBrick Secretion Tag
As our efforts to express Subtilisin E in S. Cerevisiae did not show success, we tried to create a new protein secretion tag (Mfalpha) for S. Cerevisiae as biobrick. Therefore, we used an existing fluorescence biobrick mCherry for S. Cerevisiae (BBa_E2060) and cloned it after our secretion tag Mfalpha. A scheme of the cloning can be seen below:
For more detailed labbook entries see this PDF.
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Expression of subtilisin E in E. coli
Our aim here was to express the natural subtilisin E in E. coli and to determine the optimal conditions.
Building the expresion system
Day 1
Our aim was to create a functional expression system for subtilisin E in Escherichia coli. At first, we had to optimize the sequence of the subtilisin E gene from Bacillus subtilis for Escherichia coli codon usage. For this purpose, our DNA sequence was improved with the DNA and protein sequence analysis tool ‘Geneious’ and the ‘Codon Optimization Tool’ from IDT. In the following, we proceeded to design the expression system digitally.
The expression system consists of commonly used BioBricks:< br/>
Testing the Expression
Testing the folding and activity
Mutating the expression system
Site-directed mutagenesis
Testing the expresion
Testing the folding and activity
Photoclevage
Irradiation
Testing the Expression
Testing the folding and activity
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Research on screening system. Decision to work with kit from iGEM Team Austin, Texas 2014
Definition of incubation conditions
Arrival and Preparation of Testkit with DNA from Texas
Recovery of plasmids in TRIS buffer pH 8.5, followed by transformation in BL21 DE3 gold, LB solid plates with Gentamycin 30µg/ml.
Determining concentration of antibiotcs in LB
The growth of E.coli cells transformed with plasmids wild type tyrosine -tRNA/synthetase and oNBY-tRNA/synthetase on LB solid as well as in LB liquid is not affected by concentrations of gentamycin up to 30µg/ml, whereas a wild type BL21 DE3 gold is affected already at 5µg/ml. Hence, appropriate concentration for gentamycin plates and cultures is 30µg/ml. This observation is also valid for 50µg/ml Kanamycin tested with transformed reporter plasmids in BL21 DE3 gold.
Furthermore the combination of the two antibiotics is evaluated. Since BL21 DE3 gold showed a proper growth rate in LB with 30µg/ml gentamycin + 50µg/ml Kanamycin when transformed with a reporter plasmid and a synthetase plasmid the assessed concentrations are kept.
Determining concentration of atibiotics in M9 minimal medium
Competent cells already containing the reporter plasmid pFRY are beeing transformed with the PCR product from Site Saturated Mutagenesis 3 onto M9 solid with different antibiotics concentration and afterwards picked in overnight cultures with the same antibiotics concentrations. A gel electrophoresis as well as a colony PCR is made to detect, if the concentration of both antibiotics is high enough to have an appropriate pressure on the cells, but is low enough, so that cultivation of cells is after all possible in minimal medium. The growth on microtiter plates is as well evaluated via plate reader.
Gentamycin 5µg/mg + Kanamycin 50µg/ml is the only concentration where E.coli is growing and reliably keeps both plasmids. As after transformation on M9 solid with Gentamycin 5µg/ml some wild type BL21 colonies grew, gentamycin concentration in M9 solid was increased: Transformation onto Gentymacin 10µg/ml + Kanamycin 50µg/ml and subsequent picking in liquid M9 cultures with Gentymacin 5µg/ml + Kanamycin 50µg/ml resulted in practically no growth of BL21 wild type but in appropriate growth rates of cells transformed with two plasmids.
- Gentamycin 10 µg/ml
- Kanamycin 50 µg/ml
- Gentamycin 5 µg/ml and
- Kanamycin 10 µg/ml
Determining cultivation temperature
All single and double transformed E.coli cells in LB liquid and solid show an appropriate growth rate at 37°C. Whereas evaluation of growth rates with M9 minimal medium gives better results in 30°C.
Preparation of chemical competent cells
A transformation of the reporter plasmid pRXG into BL21 DE3 gold onto LB solid with Gentamycin 30µg/ml is made. Subsequently some colonies are picked for overnight culture. Thereof cryo cultures are prepared as well as the isolated plasmids are sent for sequencing. One cryo culture is used to inoculate several overnight cultures from which competent cells are prepared via TFB-method.
Results from testing transformation efficiency
To get a maximum of colonies with transformation of the mutation library, the transformation method is tested as follows: Double transformation with two plasmids, i.e. pFRY and Y-RS, is generally lower in efficiency than single transformation. By a factor of ca. 1000 lower in efficiency to double transformation is double transformation with one plasmid and one PCR product. To have both plasmids in one cell, the best solution is transformation of a PCR product in competent cells which already contain the reporter plasmid pFRY. Colonies appear smaller in comparison to BL21, because they have two additional plasmids resulting in metabolic burden and hence a lower growth rate. Compared to double transformation, the number of cells is higher by a factor of 10. Own competent cells already containing the reporter plasmid have only minimal lower transformation efficiency than BL21 DE3 gold, when transformed with a single plasmid.
Results from testing various cultivation steps
As transformation on LB solid with subsequent picking in M9 liquid resulted in almost no growth, proper cell growth is achieved by the following method:
- Pick into M9 liquid: Masterplate, growth: 2 days at 30°C, 900rpm,
- Replicate into M9 liquid Screening plate, growth 2 days at 30°C, 900 rpm
- For positive screening:
- Induction with 100 µM IPTG
- 2mM DMNBS
- For negative screening:
- Induction with 100 µM IPTG
- For positive screening:
Cryo cultures are made from all intermediate steps.
DMNBS
DMNBS is soluble in 50% DMSO at basic pH. A stock of 50mM is prepared and used with a final concentration in M9 at 2mM resulting in a pH value within the medium solution of 7.55.
Determining growth with DMNBS
When replicating from a microtiter plate with M9 minimal medium into another with M9 and addition of both antibiotics, 100mM IPTG and 2mM DMNBS it is shown that growth of cells is affected slightly resulting in an elongated growth phase. Cultures reach their maximum cell density after 42-48h of growth.
Modelling of binding pocket of DMNBS-synthetase
Extensive research on previously observed mutation spots of different kinds of evaluated synthetases for ncAA as well as on highly conserved amino acids is made. Upon comparison of the crystal structures of EcLeu-tRNA/RS with various mutated tRNA/synthetase pairs eventually the decision is made for mutating wild type Methanoococcus janaschii tyrosyl tRNA/synthetase pair to a DMNBS specificity. Clashes of the synthetase backbone with DMNBS do not occur. Furthermore this pair had been reported to be orthogonal in E.coli. The mutation sites are determined with respect to chemical properties, space for photoprotection group and codon usage. The well working DMNBS-tRNA/synthetase pair derived from Leucyl Ec-tRNA/syntehtase pair is evaluated in order to align mutation spots.
SDM Site Directed Mutagenesis
Tyrosine 32 is mutated to glycine in a site directed mutagenesis with a full gradient two step PCR due to it is known H-bonds with former ligand of the tRNA/synthetase tyrosine. Elongation time is set to 50sec/kbp.
Gel electrophoresis shows a successful amplification at the expected length. Tubes are pooled, cleaned and transformed into E.coli BL21 DE3 gold. Sequencing results show in 4 out of 4 colonies the replacement through glycin.
(picture Gel SDM)
Initially it was tried to saturate the remaining 5 mutation sites via a two step Omnichange protocol. It was shown, that, due to the given DNA sequence and the protocol recommendations, the design of one of the primers made the execution of the protocol nearly impossible. As a conseuqence we opted for repeated Single Site Saturation Mutagenesis to reach the goal.
SSM1 Site Saturation Mutagenesis 1
As the template for the first Site Saturation Mutagenesis the isolated plasmid from transformation of SDM is used. A partially randomized codon optimized for the codon usage of E.coli within the primer is selected to establish a diversity of amino acids at the mutation site. A full gradient two step PCR with an elongation time of 50sec/kbp is performed on site L65 due to its location within the 2Å radius of DMNBS. The subsequent gel electrophoresis showed the PCR product at expected length.
(picture Gel SSM1)
Tubes are pooled and cleaned and the product is transformed into E.coli BL21 DE3 gold. Sequencing of 4 colonies shows in all 4 samples the successful mutation at the SDM1 site to various amino acids as well as the selected glycin codon at position 32.
SSM2 Site Saturation Mutagenesis 2
With the purpose of performing Site Saturation muatagenesis the colonies of SSM1 are washed off the transformation plates. The isolated plasmid is used as template for SSM2 at mutation site D158 and I159. The first site is to be mutated due to known H-bonds of the tRNA/synthetase complex with the former ligand tyrosine. Second site has a sidechain which is located within the 2Å radius of DMNBS. Primer contain two partially randomized codons with one base which is left to be unchanged in the middle. A full gradient, two step PCR is performed with an elongation time of 50sec/kbp. Subsequent gel electrophoresis shows a PCR product at the expected length. Tubes are pooled and product is isolated and subsequently transformed into E.coli BL21 DE3 gold. 4 out of 4 colonies are sequencend, where all previous mutation sites showed a glycine codon in SDM position as well as different mutations in various combinations at the three SSM sites.
(Picture Gel SSM2)
SSM 3: Site Saturated Mutagenesis 3
Prior to washing the colonies off the transformation plates it is calculated if the transformation yielded as many colonies as theoretical variants up to SSM2. As counted colonies are twice as much as theoretical variants, colonies are washed off the transformation plates from SSM2. Plasmids are isolated and used as template for Site Saturation Mutagenesis 3. A full gradient two step PCR is performed with 50sec/kbp elongation time. Gel electrophoresis is showing the PCR product to be at the expected length.
(Picture Gel SSM3)
Mutation Library
For purposes of sequencing 12 transformations of the mutation library into BL21 DE3 gold are made and all sample results show different codons in various combinations at all mutation sites, respectively a glycine at position 32.
As the mutation steps are now finished the pooled and cleaned up PCR product is transformed into the competent cells containing already the reporter plasmid, and is streaked on M9 solid with the assessed concentration of antibiotics for selection. After two days of incubation appropriate colonies are picked into microtiter plates containing M9 liquid and respective antibiotics. Thus over 8000 clones are collected. Following the previous assessment, an intermediate inoculation step was made and subsequently a final inoculation into black microtiter plates with transparent bottom. Induction with 100µM IPTG as well as supplementation of 2mM DMNBS was carried out at the beginning of the cultivation (see results).
(Picture of diversity of library, screenshot Geneious) .
Determine measurement conditions and parameter
Screening
Analysis
