Team:Bielefeld-CeBiTec/Notebook/CFPS
Cell-free Protein Synthesis
04/22: Transformation of electrocompetent E. coli KRX with BBa_K1365020 (sfGFP (Bs))
04/23: preparing glycerol stock of KRX with BBa_K1365020
04/24-04/25: Mini-prep of BBa_K1365020
04/28:
- Transformation of electrocompetent E. coli KRX with BBa_I746909 (T7-promoter, RBS, sfGFP, double terminator). Before plating on LB(Cm), 50 µl of 200 g∙L-1 Rhamnose-solution is added to the plate to induce T7-Polymerase. Inoculation of 10 ml preculture of simple KRX from plate. Overnight culturing, shaking vigorously at 220 rpm.
04/29:
- colonies of KRX with BBa_I746909 look greenish! 2.2 ml Overnight cultures from KRX are used to inoculate 100 ml for start OD600=0.144. Measuring of growth curves for a first estimation of appropriate moment for cell harvest (see image below)
Growth curves (duplicate) from E. coli KRX at 37 °C in 100 ml 2xTY+P-medium. 1 l shaking flasks without baffles were used. Shaking at 200 rpm.
04/30:
- An aliquot of ER2566 also known as "T7 Express Competent E. coli (High Efficiency)" from NEB was kindly provided by Fabian. Cells were plated on LB.
- Plasmid-mini-prep from 3 ml of overnight culture (E. coli KRX with BBa_I746909), two glycerol stocks prepared.
- Preparation of amino acid stock solutions for CFPS acccording to protocol from Caschera and Noireaux 2015. First, all aminoacids were seperatly weighed into microcentrifuge tubes, which took a while. Then, 500 µl of 5 M KOH was added to each amino acid. Solubilization was achieved via multiple inverting and, if necessary, vortexing. Especially tyrosine takes a while, and is a suspension rather than a solution. Stock solutions are afterwards stored at -20 °C. Note: According to Caschera and Noireaux, these stock solutions can only be stored a few weeks.
05/04:
- Preparation of two equimolar amino acid mixtures à 381.6 µl according to Caschera and Noireaux 2015 (see protocols). Water is added to a final volume of 4 ml each. pH adjusted with 110 µl glacial acetic acid to pH = 6.5 and aliquoting in 0.5 ml Eppendorf-tubes à 410 µl and 205 µl, respectively. Aliquots were flash-freezed in liquid nitrogen and stored at -80 °C.
- EcoR1 and Pst1 restriction analysis of isolated plasmid with BBa_I746909, bands as expected.
- 2x 10 ml 2xYT+P medium are inoculated with ER2566 from plate. 1x 10 ml is inoculated with KRX from glycerol stock. All cultures grow over night in 100 ml shake flasks at 37 °C and 220 rpm.
- 05/05: Inoculation of 3x 100 ml 2xYT+P, each with one of the overnight cultures. Start OD600:
- KRX ⇒ 0.139
- ER2566 No. 1 ⇒ 0.146
- ER2566 No. 2 ⇒ 0.140
- ⇒ Plotting of growth curves. After 1.75 h, KRX and ER2566 No.2 are induced with 0.5 ml of 200 g∙L-1 Rhamnose and 100 µl of 0.5 M IPTG to observe if production of T7-Polymerase has an impact on growth behavior.
Growth curves of ER2566 and KRX
Also, Primerdesign for RraA and RraB.
05/06: Preparation of ON cultures:
- T7p-RBS-sfGFP in 10 ml LB with Cm and 50 µl of 20% (w/w) Rhamnose
- T7p-RBS-sfGFP in 25 ml LB with Cm for midi-prep
- Rosetta-gami2 in 10 ml 2xYT+P for growth curve
05/07:
- Proving expression of sfGFP when T7-Polymerase is expressed in KRX through addition of Rhamnose
- midi-prep of 25 ml T7p-RBS-sfGFP ON culture
- measuring of growth curve of Rosetta-gami2 (conditions like 05/05)
Growth curves of Rosetta-gami2 in 100 ml 2xYT+P. Similar to 04/29, 1 l shaking flasks without baffles were used. Shaking at 200 rpm.
05/11:
- Preparation of S30-Buffer (10 mM TRIS, 14 mM Mg-Glutamate, 60 mM K-Glutamate, to pH = 8.2 with acetic acid), autoclave
- Inoculation of 10 ml 2xYT+P with ER2566 from glycerol stock, ON culture
05/12:
- Inoculation of 2x 200 ml 2xYT+P (in 1 l shaking flasks without baffles) with ON culture of ER2566 to start OD600 = 0.137, 200 rpm, 37 °C
- Cultures induced at t = 1.5 h with 200 µl of 0.5 M IPTG each for a final IPTG concentration of 0.5 mM. For OD600 progression see the following table.
| time | OD600 flask 1 | OD600 flask 2 |
|---|---|---|
| 0 h | 0,137 | 0,137 |
| 1 h | 0,379 | 0,397 |
| 1.5 h ⇒ induction | 0,866 | 0,838 |
| 3.5 h | 2,405 | 2,446 |
| 4 h | 2,657 | 2,585 |
- Cell harvest (see protocols)
05/13:
- Cell pellets thaw on ice for 1.5 h. Pellet weight: 0.677 g and 0.655 g respectively
- Cells are resuspended in 1.5 ml icecold S30-buffer (with 3 µl 1 M DTT added each) with 10s vortex - 30 s ice resting cycles until pellets are fully resuspended. Suspensions are each transferred to a 2 ml microcentrifuge tube.
- Cell disruption via sonification. Our power calibration (see protocols) revealed that the energy input of this specific Sonificator (Bandelin HD2070, MS73 tip) at this specific setup (70% power, cycle = constant) was about 3,69 J∙s-1. Therefore, for a total energy input of about 900 J, 24x 10s sonification - 10s cooling cycles were performed. The tip was dipped in about 2/3 of the cell suspension. During sonification, the microcentrifuge tube rested in ice rather than in an ice bath. As we observed that the tube becomes slightly warmer in the first case, all later sonifications were conducted with an ice bath.
- After 10 cycles of sonification / cooling, we took 10 µl of the first tube after each further cycle and diluted them in 990 µl each. These samples were later used to conduct our first test about how effective our sonification kills E. coli: 20 µl of the diluted samples were plated on LB
- Last steps of cell extract harvest were carried out as described in the protocol section with following variation:
- cell extract in tube 2 has to be centrifuged a second time as the unsolvable fraction did not precipitate completely after the first centrifugation
- 10 µl of cell extract (supernatant after centrifugation) of tube 1 are separately flash-freezed for subsequent Bradford-Assay
- Cell extract from tube 2 is subjected to a run-off reaction for 50 min at 37 °C at 300 rpm in a thermomixer.