iGEM Bielefeld 2015

Heavy Metals

Week 1 (4/20/2015-4/26/2015)

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.

Week 2 (4/27/2015-5/3/2015)

We transformed BioBricks from the distribution:

BBa_K346001(MerR) without success
BBa_K346002(PmerT) without success
BBa_K608002(konstitutiv Promoter+RBS) without success

Week 3 (5/4/2015-5/10/2015)

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.

We repeat the Transformation of BioBricks from the distribution:

BBa_K346001(MerR) without success
BBa_K346002(PmerT)
BBa_K608002(konstitutiv Promoter+RBS)

Colonies were used to inoculate overnight cultures.
Plasmids were isolated using a miniprep kit.

Gibson-Primerdesign

Week 4 (5/11/2015-5/17/2015)

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Week 5 (5/18/2015-5/24/2015)

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Week 6 (5/25/2015-5/31/2015)

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Week 7 (6/1/2015-6/7/2015)

phuisopn PCR with temerature gradient between 55-63°C

with pSB1C3_rcnR_fwd/rev (calulated annealing temperatures 66°C and 71°C
with pSB1C3_copAP_fwd/rev (calulated annealing temperatures 53°C and 73°C

PCR wordked, positive control worked, no amplification of copAP and rcnR.

Week 8 (6/8/2015-6/14/2015)

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.

Week 9 (6/15/2015-6/21/2015)

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.

Week 10 (6/22/2015-6/28/2015)

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.

Week 11 (6/29/2015-7/5/2015)

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.

Week 12 (7/6/2015-7/12/2015)

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.

Week 13 (7/13/2015-7/19/2015)

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:

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.

Week 14 (7/20/2015-7/26/2015)

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.

Week 15 (7/27/2015-8/2/2015)

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.

Week 16 (8/3/2015-8/9/2015)

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.

Background fluorescence of arsenic devices

Week 17 (8/10/2015-8/16/2015)

Week 18 (8/17/2015-8/23/2015)

Optimization of arsenic repressor for CFPS

In order to thest whether it is possible to build a CFPS biosensor by co-expressing the repressor protein in the reaction, the his-tagged arsenic repressor plasmid was optimized.
A double terminator was inserted by 3A assembly. However, a test digestion showed unexpected bands.
Sequencing confirmed that the double terminator was in fact a different BioBrick.

Week 19 (8/24/2015-8/30/2015)

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.

Week 20 (8/31/2015-9/6/2015)

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 cultures 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 were measured in a Tecan Reader.
The OD₆₀₀ of LB medium was subtracted from the other OD values. The RFU was then divided by the corresponding OD and the average of the normalized RFU of the arsR cultures was subtracted from each value.

Week 21 (9/7/2015-9/13/2015)

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Team:Bielefeld-CeBiTec/Notebook/HeavyMetals

Heavy Metals