Split IFP & GFP  - Week 1

1        CPXR extraction

Extraction of CPXR from genome of E. Coli strain K-12 MG1655

1.1       Material and methods

NEB Phusion HF PCR Protocol (see protocol), with corresponding primers (cf primers datasheet) for amplification. 

1.2       Results

1.2.1     Data

1.2.2     Interpretation

CPXR expected size : 699 bp

Amplification of CPXR worked as expected.

2        PCR for amplification of iGEM plasmid pSB1C3 and for addition of overlapps to CPXR

Amplification of pSB1C1 backbone from Cambridge biobrick K325219. This backbone contains the RBS, an arabinose-inducible pBAD promoter, iGEM prefix (containing ECORI and xbaI restriction sites) and suffix (contaning SpeI and PstI restriction sites), a chloramphenicol resistance gene, and an origin of replicaiton. Additon of overlapp to CPXR in order to make a Gisbon assembly of the backbone and the CPXR

2.1       Material and methods

NEB Phusion PCR and suitable primers.

2.2       Results

2.2.1     Data

2.2.2     Interpretation

We obtained the expected amplicon, and are now able to make a Gibson assembly to fuse the backbone amplicon and the CPXR.

1      Gibson assembly and colony PCR of pCPXR

Fusion of pSB1C3 backbone and CPXR

1.1       Material and methods

Gibson assembly kit (see protocol) for the Gibson assembly, and Taq polymerase for colony PCR (see protocol)

1.2       Results

1.2.1     Data

Colonies were obtained and analyzed by colony PCR, usiong VF2 and VR primers form iGEM

1.2.2     Interpretation

The amplicons have the expected size, meaning the Gibson assmbly worked ant that we very likely obtained pCPXR (plasmid with pSB1C3 backbone and CPXR). Sequencing will reveal if the plasmid contain any mutation.

2        Sequencing of pCPXR

Confirmation of pCPXR sequence with Microsynth sequencing services.

2.1       Material and methods

We sent the plasmid with a concentration of 80 ng/μl and with premixed primers VR and VF2

2.2       Results

2.2.1     Data

We obtained the right sequence, excepted one deletion of a cysteine in the ara promoter at the position -41. This deletion appeared to be originally in the backbone pSB1C1 that we amplified from Cambridge biobrick K325219, without consequence on the transcription on the coding sequence.

3     Amplification of IFP1, IFP2 and pCPXR with suitable overlaps to fuse the split at the C or N terminal of CPXR

PCR amplification of fragments to fuse IFP1 and IFP2 at the C or N terminal of CPXR in pCPXR plasmid

3.1       Material and methods

NEB Phusion kit (see protocol) and suitable primers (see list). Template come from Michnick lab IFP PCA.

3.2       Results

3.2.1     Data

3.2.2     Interpretation

IFP1 C didn't work but the other ones have the expected size. We are going to try again IFP1 C in a gradient PCR

1        Colony PCR for IFP2-pCPXR (at the C and N terminal)

To check if colonies were transformed with our GA product (pCPXR fused with IFP2 at the N ter and C ter) or with empty plasmid (pCPXR without the insert).

We did the GA with PCR product of pCPXR opened with corresponding primers and amplified insert, but we didn’t get rid of the pCPXR DNA template so competent cells may have been transformed with empty vectors thus acquiring chloramphenicol resistance. This is why we obtained so many negative colonies.

1.1       Material and methods

We used VR ans VF2 sequencing primers.

1. pick a colony and pouf pouf in 10ul tubes of water. Directly put the tip in 3ml of LB with chloramphenicol and grow the culture.
2. Boil the 10ul tubes of water containing the picked bacteria at 95° for 10 minutes. It will be your « DNA template ».
3. Prepare the PCR mix for Taq polymerase, with 1ul of DNA template

1.2       Results

1.2.1     Data

Colonies that are thought to contain the insert IFP2 are squared. Red means that IFP2 is at the N terminal and blue at the C terminal. We made a glycerol stock of these cells and mini-prep

1.2.2     Interpretation

By observing the difference of size between the negative control (PCR of a vector that doesn’t contain the insert) and the colony PCR fragment, we see that we obtained some colonies containing empty vectors and some that contains very likely IFP2 insert. Further IFP2 and sequencing will confirm it.

2        PCR amplification of IFP2 in the plasmid supposed to contain IFP2

Amplification of IFP2 from IFP2-pCPXR plasmid, to confirm that our GA product contains the insert IFP2.

2.1       Material and methods

We used primers previously used to amplify and to add an overlap to IFP2 Cter and Nter respectively. As the « tail » of the primers were now annealing the plasmid, one could think that changing the Annealing temp was necessary but you can keep the same that you used the first time (Ekaterina). Unfortunately some of the PCR tubes opend in the PCR machine so we couldn’t analyzed all of the plasmid.

2.2       Results

2.2.1     Data

2.2.2     Interpretation

All of the plasmids analysed contain the insert IFP2 and the size is correct (600 bp). Our GA worked, at least for the colonies that were analyzed on this gel. We are now ready for sequencing

3        Sequencing IFP2-pCPXR plasmid

To check if our GA products, pCPXR fused to IFP2 at its Cter or Nter don’t contain any mutation.

3.1       Material and methods

We send for sequencing the plasmid from the colony 4 red, corresponding to plasmid pCPXR-IFP2 at the Nterminal.

3.2       Results

3.2.1     Data

IFP2 is intact. Unfortunately CPXR contains one mutation, a A instead of a G at the base 2516, changing a glycine ggt to a       gat. We don’t understand why because we previously sequenced the pCPXR and no mutation were seen. We should send an other sample for sequencing.

4        Colony PCR for IFP1-pCPXR (at the C and N terminal)

To check if competent cells were transformed by our expected Gibson Assembly product, IFP1 fused at the Nterminal and Cterminal of CPXR, and not by the empty plasmid pCPXR.

4.1       Material and methods

(See above for protocol)

WARNING : We should always do a negative control for the colony PCR with Bacteria transformed with the empty vectors, not a PCR of an empty plasmid. Otherwise you cannot make any conclusion if the colony PCR fails or if the Gibson Assembly fails. You can compare the colony PCR quality of an expected product (pCPXR) and colony PCR of your new Gibson Assembly.

4.2       Results

4.2.1     Data

5        PCR amplification of IFP1 in IFP1-pCPXR plasmid (after mini-prep)

As we coudn’t conclude with the colony PCR, we min-prep and made glycerol stock of all colonies.

5.1       Material and methods

IFP1 amplification of the obtained plasmid was done with primers previously used to add to IFP1 the overlapp (IFP1_Cter and IFP1_Nter). We used Taq polymerase.

5.2       Results

5.2.1     Data

IFP1 amplification from colonies supposed to have the plasmid pCPXR with IFP1 at its Nterminal.

Amplicon expected size is 463 bp, which correspond to our results. Colonie 5 apparently doesn’t have IFP1. 2 and 3 have a weak one. “+” means that we amplified IFP1 from the plasmid known to contain this sequence. We also obtained a band at 850 bp for all the colonies that have the IFP1 fragment. Interestingly we also obtained this band in the positive control…

IFP1 amplification from colonies supposed to have the plasmid pCPXR with IFP1 at its Cterminal.

Expected size: 484 bp which correspond to our results.

https://static.igem.org/mediawiki/2014/a/a8/20.07.14_competent_cells.pdf

Experiment 1: Arabinose promoter characterization

1.1       Material and methods

Cells with sfGFP CpxR & CpxR sfGFP constructs were cultured in different concentrations of arabinose to check the functionality of the promoter. The concentration of arabinose per sample is indicated in Table 1.

Table 1 - This table shows the concentration of Arabinose in each sample, as well as the quantity of medium and of Arabinose needed to reach it.

The negative control was the medium without cells, and the positive control was a sample of cells expressing sfGFP contitutively.

Procedure

Cells from glycerol stocks were grown in 3 mL LB selective medium (chloramphenicol) during 3h (37 ºC & shaking).

50 uL of cells were resuspended in a total volume of 5 mL of LB-chloramphenicol and Arabinose and incubated 5h at 37 ºC with shaking until an OD of ~1 was reached.
The samples were then washed twice with PBS to a final OD of 0.4 - 0.5.

Table 2 shows the quantity of PBS added to each sample (depending on the OD), as well as the OD after both PBS washes. This information is used to normalize the plate reader results.

 Table 2 - OD measurments of the samples can be seen, as well as the OD after the PBS washes.

Results

1.4.1     Data

https://static.igem.org/mediawiki/2014/0/06/Sfgfpcpxr_gradient.jpg

Figure 1 - Photo of the pelleted samples between PBS wash steps 1 and 2.

As can be seen in the image, there is a gradient of fluorescence intensity.

The results of the plate reader are depicted in Figure 2.

https://static.igem.org/mediawiki/2014/f/f5/Twotermscpxrgfp.jpg

Figure 2 - Graphs of the intensity curves of the samples. Cterm corresponds to CpxR sfGFP (sfGFP attached to CpxR's C temrinus) and Nterm corresponds to sfGFP CpxR.

1.4.2     Interpretation

As can be seen in the graph, the the results of the sfGFP CpxR sample was much better (closer to the expected results) than the one on the CpxR sfGFP, although in general, the curves grow.

It could be seen that adding too much arabinose decreases viability, so we considered that a concentration of 5 mM was the optimum arabinose concentration.