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
Determination of Parameters
Cloning
Test Expression
SDM Ser to UAG
Test Expression
Expression of Synthetase
Determination of Parameters
Check genes
Test Expression
Co-transformation
Test Expression
Test Photoclevage and Activity
New BioBrick Secretion Tag
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Expression of subtilisin E in E. coli
Building the expresion system
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 antiobiotcs 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 a 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: Transformatiom 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.
Preparing 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. Therof 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 oft he 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:
- nduction 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 muatagenesis with a full gradient two step PCR due to ist known H-bonds with former ligand of the tRNA/synthetase tyrosine. Annealing 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 4 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 anneanling 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 show 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 plasmid is isolated and 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 annealing temperature 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 annealing 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
