Template:Team:Edinburgh/Notebook/Week03

1)Transformation of Citrobacter with various replicons (results)


These results show that all but one of the plasmids have successfully been transformed into both E. coli and Cf, therefore these replicons are compatible with Cf. Cf cells with the multi-host plasmid did not grow at all, and there can be several reasons why: the codon usage in Cf differs from E. coli and Bacillus; the promoter does not work properly in Cf or Cf is less resistant to chloramphenicol. Bacteria transformed with ptg262 will be plated onto plates with differing concentrations of chloramphenicol to assess whether this plasmid is compatible at all with Cf.
The pSB2K3 transforms were plated onto plates containing X-gal in order to assess whether the Cf cells were lacZ positive (had a lacZ gene on their chromosome). Since the colonies turned blue, this means that the Cf cells are lacZ positive (as the plasmid contained no lacZ minigene) whereas the E. coli cells are lacZ negative since the colonies were red. The RFP was expressed in both cells, as evidenced by red fluorescence under a blue light.

2)LA agar plates with different metronidazole and DNBA concentration were examined.

No IPTG was added and no difference was seen between the strains. However, 50 ul (0.1 mg/ml) DNBA/met was chosen as further concentration to use since it did not seem to inhibit growth of the strains substantially.

3)Repeating LA agar plates with metronidazole, nitrofuratoin and DNBA.

50 mg/ml stock of nitrofuratoin ( antibiotic toxic to E.coli) was prepared.
Plates with 0.1 mg/ml DNBA, met or NFT plus and minus IPTG were prepared and inoculated with the four nitroreductase strains.
4)Test if sucrose hydrolase can act as arsenic detector

Chloramphenicol (5 ul out of 40 mg/ml stock), sucrose ( 250 ul out of 20% sucrose stock) and 0/5/25/50 ul As (out of 10 000 pps As stock) were added to LB (5 ml). The four bottles were inoculated with pSBIC3-J33207 CScA blue (4)

5) PCR for MtrCAB and ccmA-H

Stock solutions of primers were prepared by adding water to fresh primers in order to reach 500 pmol/ul concentration.
Working solution was prepared by adding water to stock solution in order to reach 10 pmol/ul concentration.
Cell suspensions of E. coli and S. oneidensis were obtained by inoculating cells in 150 ul water
PCR mixture was prepared according to Cfrench:PfuPCR protocol and PCR was performed for total of 100 minutes.
In the meantime agarose gel was prepared following Cfrench:AGE protocol
PCR product was tested on the gel resulting in clear band of about 6 kb for ccm and no bands for Mtr


ccm PCR product was purified following Cfrench:DNAPurification1 protocol . Purified product was left in freezer for storage [freezer 1 (B grade freezer, the leftmost one), orange rack on top shelf, 1st row, 5ft tube markes KK 9.7.12 ccm PCR E. coli]

1) Cloning of ccm PCr product into vector

pSB1C3 vector with RFP was used
PCR product was cloned using modified Cfrench:bbcloning protocl
reaction mixture:
water 30 ul
vector pSB1C3 5 ul
PCR ccm 5 ul
buffer 3 5 ul
BSA 1 ul
XbaI 2 ul
PstI 2 ul
components were mixed, pulse-spinned in a centrifuge and incubated for 3 hours in 37 oC

Vector cloning was followed by DNA purification (protocol) and ligation by adding following reagents to purified mixture:
dH2O 7 ul
ligase buffer 2 ul
T4 ligase 1 ul
Sampels were incubated in 16 C waterbath overnight

2) Lac promoter characterization

Aim: To test the lacZ promoter by measuring the fluorescence of the RFP gene tied to this promoter. The reason for doing this is because it is not yet known whether our strain of Cf has a lacI gene. If the lacI gene is present on the host’s chromosome, we expect the fluorescence to be much lower in the NO IPTG bottles than in the ones containing IPTG as the promoter will not be on. If there is no lacI gene, we expect the fluorescence in all 3 bottles will fall into a similar range.
Method: In order to characterize the Lac promoter in Cf, 5ml LB bottles were inoculated with colonies from the E. coli/Cf + RFP plates made on Friday. 5 uL of chloramphenicol40 were added to each of the bottles and varying amounts of IPTG were also added, as is shown in the table below. The bottles were then incubated in the warm room on the shaker.


3)Multi-host vector characterization in Cf

Aim: To assess whether the multi-host vector BBa_I742123 is compatible with our strain of Cf, as there was no growth on the relevant Cf plate made on Friday, while there was some growth on the E. coli plate. One reason for this may be that the Cf cells are less resistant to chloramphenicol than E. coli, so we are plating them onto plates with varying concentrations of chloramphenicol to see whether they grow at all.
Method: 500ml of LB was microwaved for 4+4+4+2+1 (15) minutes and then placed in a 55C waterbath to cool. After an hour, 500uL of IPTG90 was added to the bottle and the agar was poured on plates containing varying concentrations of chloramphenicol40, and, after the agar set, loopfulls of the transforms made on Friday were streaked onto the plates, as is shown in the table below. The pSB2K3-containing (and therefore kanamycin, not chloramphenicol resistant) cells were used as controls to test background growth in varying amounts of chloramphenicol. All the plates were then incubated at 37C.


4)LA plates with MTZ, DNBA and NFT were examined.

No substantial growth was seen on any of the NFT plates probably because these concentrations were too high. BS-contol, nitred-6, CFX-nitred showed some growth on DNBA plate however BS-nitred grew poorly. Other strains grew on the MTZ plates while BS-nitred did not grow at all in +IPTG plate. On MTZ – IPTG plate, BS-nitred was growing. Contol plates showed that BS-nitred grew poorly in the presense of IPTG probably due to metabolic stress.
http://s1056.photobucket.com/albums/t366/edigem12/100712/
The experiment was repeated with 50 µg/ml (25 µl out of 50 mg/ml stock) and 70 µg/ml (35 µl out of 50 mg/ml stock) of DNWA/MTZ plus IPTG (25 µl out of 90 mg/ml stock). In addition, 2/6/10 or 20 µg/ml NFT ( 1/3/5/ or 10 µl out of 50 mg/ml stock) plates + IPTG were prepared. Control + and – IPTG was also prepared. All plates were inoculated by the four nitroreductase strains. All plates were left o/n at 37ºC.

5)Sucrose hydrolase arsenic detector test (prepared on 9th of July)

The pH of all liquid cultures was measured:
0 ppb As 6.30
10 ppb As 6.26
50 ppb As 6.04
100 ppb As 5.84
The experiment was repeated however lactose was used instead of sucrose. The liquid cultures were left at 37ºC with agitation o/n.

1) Lac promoter characterization - results and further tests

The cultures all showed some growth but the E. coli + 2 μL IPTG was the only culture that turned red. The fluorescence of all the cultures were measured, using a green filter, and the results for Cf and E. coli can be seen in the graph and table, respectively, below.

These results show that E. coli needs IPTG to start producing the RFP gene, as the LacZ promoter is inhibited by lacI in the absence of IPTG. There is no significant difference between the amount of fluorescence in Cf cells that were grown in the absence, or in the presence of 2 2 μL IPTG, which can mean that the promoter is not inhibited even in the absence of IPTG, but that higher amounts of IPTG may help transcription.
Further tests: Bottles containing 2.5ml LB + 2 μL chloramphenicol (no IPTG) were inoculated with 20 μL transformants from the E. coli / Cf + RFP tubes and put in the warm room on the shaker. The OD of these cultures will be measured tomorrow morning and normalized (by diluting the higher OD culture to match the lower OD) in order to ensure that we start with the same amount of cells. These will then be transferred to bottles containing 2.5ml M9 medium, 2.5 μL chloramphenicol and varying concentrations of IPTG (No IPTG/1-5μL IPTG), and left to grow overnight. The reason for this is that M9 will have less background fluorescence than LB and the fluorescence will arise from the same number of cells in each bottle.

2) Multi-host vector characterization in Cf - results

No growth was observed on any of the plates except the control plate with 5 μL chloramphenicol, this is likely to be background growth. The plates were left in the incubator for a further day.

3) Arsenic detector test liquid sucrose ( prepared on 9th of July 2012) and lactose (prepared on 10th of July 2012) cultures were examined.

Both pH and OD600 with LB blank were measured:
Sucrose – pH – OD600
0 ppb As – 6.07 – 1.339
10 ppb As – 5.68 - 1.507
50 ppb As – 5.63 – 1.540
100 ppb As – 5.50 – 1.619
Lactose – pH – OD600
0 ppb As – 6.41 – 1.319
10 ppb As – 4.94 - 1.426
50 ppb As – 5.15 – 1.322
100 ppb As – 5.01 – 1.431

4) LA plates with MTZ, DNBA and NFT (prepared 10th July) were examined:

Control -/+ IPTG showed good growth
NFT:
20 µg/ml – no growth
10 µg/ml other cultures showed some growth while BS-nitred did not grow.
2 µg/ml and 6 µg/ml – all strains showed some growth
MTZ
50/70 µg/ml – all strains showed some growth
DNBA
50/70 µg/ml – all strains showed some growth. BS-nitred grew poorest.
MTZ ( from 9th July 2012)
BS control grew fine, nitred-6 and CFX-nitred did grow pooly and BS nitred did not grow at all.
http://s1056.photobucket.com/albums/t366/edigem12/110712/
M9 plates medium was prepared and left for autoclaving.


5) ccm transformation
Following the ligation, competent E coli cells were transformed with the plasmid (protocol

1)The M9 agar was prepared with 1.5 % (1.5 g agar in 100 ml). Plates with 400 µl lactose/sucrose/glycerol/glucose or no sugar were poured. They were dried and sealed with parafilm and left in the cold room over the weekend.

Cell extract preparation
Cells from cytochrome expression cultures from Week 2, 06.07.2012, Friday were used to obtain cell extract by following the protocol:
5 min centrifugation at 10k rpm
supernatant discarded, pellet resuspended in 250 ml PBS, the resuspended mixtures were combinded to give 500 ml total of each culture
samples were kept on ice and sonicated (6 cycles of 10s sonication and 20s recovery)

PCR for MtrCAB
PCR was performed for MtrCAB gene cluster using two potential S. oneidensis strains as template ( protocol)

1) Multi-host vector characterization in Cf - results

Very little growth was observed on any of the plates, so they were put back in the incubator for a further day. It is likely that the plasmid does not function properly.

2) Preparation of M9 plates to characterize Citrobacter and E. coli growth on various sugars
M9 agar was prepared yesterday and the bottle autoclaved, 3.4ml thiamine hydrochloride (10mg/ml) was added to the liquid agar (at 55C) today and then the agar was poured onto 5 plates: no sugar, sucrose, glucose, lactose and glycerol. The agar was left to set and stored in the cold room overnight, the plates will be streaked tomorrow with 2 strains of Cf, E. coli and E. coli with the sucrose hydrolase gene.


3) Transformation results
Subcultured the transformed cells that lost RFP to separate plate.