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.

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:

1. Pick into M9 liquid: Masterplate, growth: 2 days at 30°C, 900rpm,
2. Replicate into M9 liquid Screening plate, growth 2 days at 30°C, 900 rpm
   1. For positive screening:
      1. Induction with 100 µM IPTG
      2. 2mM DMNBS
   2. For negative screening:
      1. nduction with 100 µM IPTG

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.