Team:Bielefeld-CeBiTec/Notebook/HeavyMetals
Heavy Metals
- We transformed BioBricks from the distribution:
- BBa_J33201 (arsR)
- BBa_K516030 (mRFP1 with RBS and terminator)
- Colonies were used to inoculate overnight cultures.
- Plasmids were isolated using a miniprep kit.
- ArsR (BBa_J33201) and RFP (BBa_K516030) were combined using 3A assembly:
- Restriction digest of arsR, RFP and pSB1K3.m1
- PCR Clean-up
- Ligation: 2 µL per fragment, 30 min
- Transformation by electroporation and heat shock (for comparison)
- No colonies, therefore the ligation was repeated with significantly more DNA. Incubation overnight.
- Electroporation resulted in colonies, which were screened with a colony PCR.
- A product length of 1705 bp was expected. The gel shows that most plasmids seem to be correct.
- This result was confirmed by sequencing.
- Insertion of a second repressor binding site into BBa_J33201
- Two primer pairs (Cm_fwd, Ars_1_rev, Ars_2_fwd, Cm_rev) were used to amplify the plasmid and introduce a second repressor binding site after arsR
- A PCR with Phusion Polymerase failed. A gradient PCR did not show any improvement.
- A comparison of Phusion and Q5 showed that Q5 works very well.
- However, an upscalling of the reaction to produce more product did not work.
- Therefore, two 20 µL reactions were used per fragment. This PCR worked well and the products were extracted from the gel.
- We performed a Gibson assembly reaction with the two fragments and transformed the mix via heat shock.
- A colony PCR was performed with five colonies, but only one reaction yielded product.
- Five colonies were used to inoculate overnight cultures.
- Plasmids were isolated from the cultures and a restriction digest was performed to check them.
- The bands were as expected for all colonies.
- One plasmid was handed in for sequencing, which confirmed its correctness.
- 3A assembly of the modified arsR and RFP (BBa_K516030)
- Restriction digest of the modified arsR with EcoRI-HF and SpeI-HF
- Heat inactivation: 20 min at 80 °C
- Ligation with RFP and pSB1K3 (from week 3)
- Electroporation with 2 µL ligation mix
- Only one colony grew on the plates, which according to a colony PCR was not correct.
- 5 and 20 µL of the ligation mix were transformed again into chemocompetent cells.
- This time there were plenty of colonies. 10 were screened with a colony PCR and most appeared to be correct.
- First test with arsenic sensor
- 5 ml cultures (LB + kan) were prepared from glycerol stocks of E. coli harboring pSB1K3-arsR-RFP and pSB1K3-arsR-arsO-RFP and cultured at 37 °C.
- After 16 h, cultures were diluted 1:100 with fresh LB + kan.
- After 1 h at 37 °C, arsenic at 1000 µg/L was added.
- After 24 h at 37 °C, the cultures were centrifuged (5 min, 5000xg).
- The cell pellets were clearly red, indicating that the arsenic sensors are functional.
- Arsenic sensor test in 96 well plate
- Dilutions of a 50 mM sodium arsenite solution were prepared
- As for the first test, 5 mL cultures of the two strains were inoculated from glycerol stocks.
- After 18 h, the cultures were diluted to reach an OD600 of 0.4.
- After 30 min at 37 °C, 100 µL culture and 100 µL sodium arsenite solution were combined in a black 96 well plate.
- The final arsenic concentrations ranged from 0 to 1000 µg/L.
- Every combination of strain and concentration was measured in triplicate.
- The plate was incubated at 37 °C for 4 h.
- RFP fluorescence was measured in a plate reader.
- No increase of fluorescence with the arsenic concentration was observable.
- Measuring different dilutions of an RFP culture supernatant showed that the linear range was very small. Therefore, this instrument does not seem to be suitable for RFP measurements.
- Optimization of arsenic sensor for CFPS
- We decided to modify the arsenic sensor so that it is likely to work better in the CFPS.
- The devices we decided to clone are:
- An sfGFP under the control of the T7 promoter and the arsenic operator including the UTR which, as we showed, improves the sfGFP expression in the CFPS.
- An sfGFP with the UTR behind arsR and its natural promoter.
- A his-tagged arsR for purification and controlled addition to the CFPS reaction.
- In the first step, the T7 promoter needed to be inserterd in front of arsR. Therefore, BBa_I719005 (T7 promoter) from the distribution was transformed by heat shock.
- A colony PCR showed bands which matched the expected length.
- Overnight cultures were inoculated with the colonies and plasmids were isolated.
- Restriction digest:
- pSB1A2-pT7 with EcoRI-HF and SpeI
- pSB1K3-arsR-RFP with XbaI and PstI
- pSB1C3 (linearized) with EcoRI-HF, PstI and DpnI (+ FastAP)
- Ligation of the tree fragments and heat shock transformation resulted in small colonies, which were not correct according to a Colony PCR.
- Optimization of arsenic sensor for CFPS
- The ligation of T7 promoter, arsR and pSB1C3 was repeated, and this time there were several colonies which showed the expected band after a colony PCR.
- Several PCRs were performed to create fragments for Gibson Assembly:
- Ars_3_fwd and Cm_rev on BBa_J33201
- Cm_fwd and Ars_3_rev on BBa_J33201
- Cm_fwd and UTR_noT7_rev on BBa_I746909
- UTR_fwd and Cm_rev on BBa_I746909
- All reactions worked very well and the products were extracted from the gel.
- The products from PCR 1+2 and 3+4 were combined by Gibson Assembly.
- Following heat shock transformation, plenty of colonies grew on the plates.
- Some were checked by Colony PCR and all seemed to be correct.
- 3 overnight cultures per construct were inoculated and plasmids were isolated.
- The isolated plasmids were used for further cloning steps:
- A restriction digest was performed with the isolated pSB1C3-UTR-sfGFP (XbaI and PstI) and pSB1K3 (EcoRI-HF, PstI, DpnI, FastAP)
- Ligation:
- arsR-arsO2 + RFP + pSB1K3
- arsR + UTR-sfGFP + pSB1K3
- PCR:
- UTR_arsO_rev and Cm_fwd on pSB1C3-pT7-arsR
- UTR_fwd and Cm_rev on BBa_I746909
- The products were extracted from the gel and combined via Gibson Assembly.
- The Gibson Assembly and ligation mixes were transformed via heat shock.
- Optimization of arsenic sensor for CFPS
- The colonies which had grown after Gibson assembly and transformation were checked with a colony PCR. The bands were as expected.
- Overnight cultures were inoculted and the plasmids were isolated and handed in for sequencing.
- The sequencing results for arsR-UTR-sfGFP were slighly unclear at one position, but likely the device is correct.
- All other devices were clearly correct.
- Cloning of a his-tagged arsenic repressor
- To get an arsenic repressor which can be purified and added to the CFPS, a his-tagged version under the control of the T7 promoter was cloned.
- The first step were the following PCRs:
- arsRhis_arsR_fwd and arsRhis_arsR2_r on pSB1C3-arsR (BBa_J33201)
- arsRhis_Trp_fwd and arsRhis_Trp_rev on pSB1C3-tryptophanase
- The products were extracted from the gel.
- Gibson Assembly and heat shock transformation were performed.
- A colony PCR indicated that the cloning was successful, which was confirmed by sequencing.
- Expression of arsenic repressor
- An overnight culture was inoculated from the glycerol stock which had been prepared the previous week.
- Two 200 mL cultures in 1 L shaking flasks were inoculated to an OD600 of 0.1. and cultivated at 37 °C and 180 rpm.
- At an OD of 0.6, expression of the T7 polymerase was induced by adding rhamnose to a final concentration of 0.1 %.
- The cultures were grown overnight at 18 °C and 130 rpm.
- After 24 h, the bacteria were harvested by centrifugation (10 min, 5500xg).
- The cells were lysed by sonification.
- Purifiaction was carried out with the Macherey-Nagel Protino Ni-TED 1000 Packed Columns kit.
- An SDS-PAGE was done to analyze the purification.
- Measurement of the arsenic sensors´ background signal
- To reduce the background signal of the arsenic sensor, a second operator site had been introduced downstream of arsR. Due to different information concerning the exact position of the operator, two versions had been prepared.
- To find out if this had an effect, the following devices were tested:
- pSB1C3-arsR
- pSB1K3-arsR-RFP
- pSB1K3-arsR-arsO1-RFP (first version of the second operator)
- pSB1K3-arsR-arsO2-RFP (second version of the second operator)
- Single colonies were obtained from the glycerol stocks by plating.
- Five cultures per device were inoculated with single colonies.
- After 15 h, the RFP fluorescence and the OD600 were measured with a Tecan Infinite M200.
- The RFU was normalized by diving it by the OD and subtracting the singal of plain LB medium.
- The results showed that the modifications had not reduced the background signal. Instead, it was even higher than that of the unmodified device.
- Optimization of arsenic repressor for CFPS
- The UTR was inserted between the T7 promoter and arsR-his for expression in a CFPS reaction.
- A PCR was performed with BBa_K1758301 as template and the following primer combinations:
- Cm_fwd and UTR_arsR_rev
- UTR_arsR_fwd and Cm_rev
- The PCR did not work with Q5 and Phusion in HF buffer. However, it worked with Phusion in GC Buffer.
- The fragments were extracted from the gel, combined by Gibson Assembly and transformed via heat shock.
- Characterization of arsenic sensor in vivo
- Glycerol stocks of pSB1C3-arsR and pSB1K3-arsR-RFP were streaked on plates in order to obtain single colonies.
- Three overnight cultures per construct were inoculated with single colonies.
- On the following day, 50 mL culutes in LB with the correct antibiotic were inoculated to an OD600 of 0.1.
- When the cultures had reached an OD of 0.6-0.7, arsenic solutions were added to the arsR-RFP cultures to final concentrations of 0 µg/L, 10 µg/L, 50 µg/L, 100 µg/L, 500 µg/L and 1000 µg/L.
- Every 1.5-2 h, the OD600 and the RFP fluorescence was measured in a Tecan Reader.