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 2015b. 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 2015b (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.
- Also, Primerdesign for RraA and RraB.
Growth curves of ER2566 and KRX
- 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.
- Cell harvest (see protocols)
| 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 |
- 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.
- 05/20: cell disruption
- weight of the Pellet: 486 mg
- unfreeze the pellet on ice for two hours
- resuspend the pellet in 1 mL S30 buffer (with 2 mM DTT)
- 10 sec vortex
- 30 sec on ice
- repeat these steps until die pellet is resuspended (in our case we repeated these steps 5 times)
- The resuspended pellet had a volume of 1300 µL, therefore we added 200 µL S30 buffer (supplemented with 0.4 µL 1M DTT) to get a Volume of 1500 µL for the sonification step
- incubation the suspension for 10 min on ice
- sonification with Bandelin HD 2070 and the MS 73 cone end, we used 70% amplitude and no cycles
- 10 sec sonifications
- 10 sec incubation on ice
- repeat step 1 and 2 (29 times, which equals an energy of ~ 900 J/sec)
- centrifuge for 10 min, 12000 rcf
- split the supernatant into two tubes
- incubate tube 2 (650 µL) for 50 min at 37°C and 300 rpm and centrifuge again for 10 min, 12000 rcf
- aliquot the supernatant and the cell extract from tube 1 (100 µL)
- freeze in liquid nitrogen and store at - 80°C
- 05/27:
- Next CFPS test with plasmid T7p-RBS-sfGFP (BBa_I746909). Beside the negative control without DNA-template, BBa_K1365020 (sfGFP (Bs)), a plasmid with pSB1C3-backbone containing an sfGFP without promoter, is employed as additional negative control. Reactions with two different extracts (see 05/13: one with, one without run-off treatment) are performed. Furthermore, commercial RNase inhibitor is tested as it seems to be a useful supplement. A final RNase-inhibitor concentration of 1 U/µL is used in the respective reaction mixtures. CFPS is performed over night.
- 05/28:
- PCR with Phusion Polymerase, primers: pSB1C3_RraA_suf/pSB1C3_RraA_pre and pSB1C3_RraB_suf/pSB1C3_RraB_pre, template: plasmid T7p-RBS-sfGFP (BBa_I746909). No bands in agarose gel.
- Fluorescence measurement of CFPS-reaction from 05/27 with NanoDrop 3300
- Next CFPS test with plasmid T7p-RBS-sfGFP (BBa_I746909). The reactions tested are the same as on 05/27, without testing the RNase Inhibitor ⇒ glycerol may inhibit protein syntesis in vitro (Underwood et al. 2005). As a positive control, a sample from an overnight culture (BBa_I746909) induced to express sfGFP is employed. Reactions are performed at two different temperatures – 30 °C and 37 °C – in incubators over night without shaking.
- 05/29:
- No evaporation can be seen in the tubes! After 16 h incubation, the fluorescence is measured with NanoDrop 3300.
- PCR problems continue
- 06/01:
- Small amounts of amplified PCR product detectable, positive control works, but still far away from optimum.
- 06/03:
- ON culture of ER2566 (in 2xYT+P medium) is used for inoculation of 3x 100 mL 2xYT+P to OD600 = 0.13. Cultures are induced with IPTG to 1 mM final concentration after 1 h 20 min at OD600 about 0.7.
- Cell harvest at t = 3.5 h at OD600 = 3.3
- Subsequent to harvest and washing procedures, pellets are directly resuspended and lysed via sonification as before (29x for about 900 J). Half of the suspension is subjected to a run-off reaction at 37 °C for 50 min with a following . All extract is then flash-freezed and stored at -80 °C.
- 06/08:
- Primerdesign
- Preparations for next CFPS reaction
- 06/09:
- CFPS with the same templates as on 05/28
- After assembling of CFPS compounds, 10 µL of each reaction were directly pipetted into a 364 black well plate, sealed with transparent film and incubated at 37 °C. Fluorescence was measured without the film after 3, 6.25 and 24 h respectively. Fluorescence did not exceed the negative control distinctly in any case.
- 06/10:
- CFPS with plasmid T7p-RBS-sfGFP (BBa_I746909) in 21 µL volume with “normal” and run-off extracts from 05/13, 05/20 and 06/03 respectively. For each reaction a negative control with water instead of DNA is employed
- For the first time, Mg-glutamate and K-glutamate were additionally supplemented to the reactions as they were no part of the “reaction buffer”. 3 M K-glutamate and 100 mM Mg-glutamate stock solutions were prepared and directly used to supply the reaction with a final concentration of 4 mM Mg-glutamate and 80 mM K-glutamate.
- To each mix of DNA, glutamate salts and water, a master mix consisiting of “reaction buffer” and one of the above mentioned extracts was added. Reactions were performed in microcentrifuge tubes for 3 h and then 20 µL were transferred to a 364 well plate which was used for fluorescence measurements and further reaction performance. Relative fluorescence units were normalized to corresponding negative controls. The maximum of fluorescence was observed after 7.75 h for 06/03 run-off extract, which fluorescence was 10 times higher when compared to its negative control. First promising results that showed we can really produce sfGFP in vitro!
- 06/11:
- PCR with Q5-Polymerase for amplification of 5’-UTR-flanked pSB1C3-backbone. As PCR worked with Q5, most of subsequent PCRs related to CFPS were performed with Q5.
- PCR with Q5 to amplify RraA and RraB out of E. coli K12 genomic DNA and to amplify pSB1C3 backbone with RraA and RraB overhangs for Gibson assembly
- Bands as expected, gel purification of all four products. PCR for backbone amplification only worked at an annealing temperature of 71.6 °C.
- 06/12:
- Gibson assembly to insert RraA and RraB in pSB1C3 and to add UTR to T7p-RBS-sfGFP (BBa_I746909) plasmid. Subsequent transformation and plating on LB with Cm.
- Transformation of electrocompetent E. coli KRX with BBa_K864100 (SYFP2)
- 06/15:
- Glycerol stocks prepared (RraA in pSB1C3 and RraB in pSB1C3, SYFP2, T7p-UTR-RBS-sfGFP)
- Plasmid isolation of RraA in pSB1C3 and RraB in pSB1C3. Restriction analysis with Not1-HF. Bands as expected!
- Plasmid isolation of T7p-UTR-RBS-sfGFP with columns of Promega kit and elution buffer of Thermo kit (10 mM TRIS-Cl, pH = 8.5) as material ran out. Restriction analysis with Not1-HF. Bands as expected!
- Chosen of the above plasmids were send to sequencing core facility
- 06/16:
- Preparation of a new cofactor premix (see following table)
- New reaction buffer aliquots were generated in which the above mentioned cofactor premix was used. For calculation we used the excel template from Sun et al. 2013, which takes into account a reserve for pipetting.
- In subsequent CFPS-test, run-off cell extract from 06/03 was used. We tested the impact of RNase inhibitor, high amount of Mg-glutamate (12 mM) and high amount of K-glutamate (140 mM) on the reaction with T7p-RBS-sfGFP (BBa_I746909). Additionally, a T7p-UTR-RBS-sfGFP plasmid from a mini-prep plasmid isolation (06/15) was tested without any change to other components when compared to 06/10. All reactions were performed in duplicate.
- Reaction conditions and volumes were the same as on 06/10. One negative control without DNA-template was used with 80 mM K-glutamate and 4 mM Mg-glutamate added. To compare relative fluorescent units, an sfGFP-containing cell lysate and a purified GFP with 6xHis-tag (kindly provided by Lukas) was employed.
- For results, see here
- 06/17:
- Plasmid isolation of SYFP2 (BBa_K864100). Restriction analysis with Not1-HF. Bands as expected!
- Sequencing results: RraA in pSB1C3 and RraB in pSB1C3 look good, but T7p-UTR-RBS-sfGFP plasmid has only 9 A instead of the desired 10 A in spacer region. As CFPS worked well with this plasmid, it is nevertheless used in further experiments.
- Transformation of chemocompetent ER2566 with pRARE plasmid kindly provided by Julian.
| Compound | Stock in mM | volume stock solution to add in µl | concentration in cofactor premix in mM |
|---|---|---|---|
| NAD | 174 | 52.16 | 6.6 |
| folinic acid | 33,9 | 56.78 | 1.4 |
| E. coli tRNA | 50* | 110.00 | 4* |
| CoA | 65 | 114.23 | 5.4 |
| Putrescine | 200 | 137.50 | 20 |
| Hepes | 2000 | 687.50 | 1000 |
| Spermidine | 1500 | 27.50 | 30 |
| RNase-free water | to 1375 µL |
HEPES was added first so that risks due to pH changes could be averted.
* tRNA values given are in mg/mL
| Compound | Stock | Final | amount of stock for 1 buffer in µl | amount of stock for 4 buffers in µL |
|---|---|---|---|---|
| Amino Acids | 12 mM | 2 mM | 14.96 | 62.84 |
| Cofactor premix | 20x | 1x | 4.49 | 18.85 |
| NTPs | 20x | 1x | 4.49 | 18.85 |
| Na-Oxalat | 100 mM | 4 mM | 3.59 | 15.08 |
| PEP | 330 mM | 33 mM | 8.98 | 37.71 |
| Water | na | na | 1.20 | 5.03 |