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Team:UC Chile/Wet Lab
From 2013.igem.org
Wet Lab
1. Formation
To work in the expression of the Carboxysomes we modified the original plasmid pHnCBSC1D and attached a red fluorescent protein to one of the shell proteins (csoS1D) to be able to observe the Carboxysomes, this is the Carboxysome BioBrick (Bba_K1113100) + RFP (Bba_E1010). The original plasmid was already under an inducible promoter sensitive to IPTG so we could modulate the expression of the microcompartment easily.
We next tried to switch the strain and selected DH5a but this time we couldn’t regulate the expression of the RFP as the negative controls of expression shown presence of fluorescence which indicate a basal expression. For a correct assembly of carboxysomes a modulated expression it’s needed because the components of the carboxysome follow a very tight stoichiometry and the lack of this could lead to the formation of protein aggregated, to avoid this we tried to work with another strain.
Finally we selected the TOP10 strain because it showed the best expression, even though we observed basal expression of RFP the induction with IPGT enhance the presence of the fluorescence (Figure 5).
The expression of RFP on TOP10 it’s consistent with the expected on the presences of the carboxysome, the focalized pattern observed of the fluorescence along with the filamentous form of the bacteria seems to suggest the conditions for the assembly of the carboxysome.
a. Digestion of Carboxysome BioBrick (Bba_K1113100) + RFP (Bba_E1010) construct.
We digested the Carboxysome BioBrick with RFP to check that our construct were correctly assembled. The digest showed a banding pattern of the Carboxysome as expected (Figure 1) that suggested that we had correctly assembled this construct.
Figure 1. Carboxysome BioBrick (Bba_K1113100) + RFP(Bba_E1010) Gel digestión
On well 1 & 6 DNA ladder 1kb invitrogen. Well 2-5 digestion of Carboxysome BioBrick with SpeI. The expected sizes were 11090, 9256, 5738. Well 7-8 digestion of Carboxysome BioBrick (Bba_K1113100) + RFP(Bba_E1010) with SpeI. The expected sizes were 12365, 4475, 2641bp.
b. Expression of Carboxysome BioBrick (Bba_K1113100) + RFP (Bba_E1010).
To observe the expression of the carboxysome we transform several bacteria strains with CRFP and induce with 50uM of IPTG. We first started working with BL21, but as shown in figure 5 we were unable of induce any kind of fluorescence, we tried with different concentrations of IPTP (we reach 2mM!) but we couldn’t induce the expression of RFP.We next tried to switch the strain and selected DH5a but this time we couldn’t regulate the expression of the RFP as the negative controls of expression shown presence of fluorescence which indicate a basal expression. For a correct assembly of carboxysomes a modulated expression it’s needed because the components of the carboxysome follow a very tight stoichiometry and the lack of this could lead to the formation of protein aggregated, to avoid this we tried to work with another strain.
Finally we selected the TOP10 strain because it showed the best expression, even though we observed basal expression of RFP the induction with IPGT enhance the presence of the fluorescence (Figure 5).
Figure 2. RFP expression.
Different E. coli strains were transformed with Carboxysome BioBrick (Bba_K1113100) + RFP (Bba_E1010) and induced with 50uM of IPTG. The RFP expression was observed under the epifluorescence microscope with a magnification of 100X. The brightfield image and the GFP filtered are shown.
2. Targeting devices
We developed 4 different strategies to find the targeting sequence using the large and small subunits of RuBisCO linked with GFP: GFP+RuBisCO small, RuBisCO small+GFP, RuBisCO large+GFP and GFP+RuBisCO large. All this constructs were made using Gibson Assembly and were confirmed through digestion of enzymes, sequencing and epifluorescence microscopy.
After our construct was assembled, we mutated the EcoRI restriction site that was in the middle of the sequence of the large RuBisCO subunit. To do this, we used Gibson Assembly method with a mutated tail in one the primers. We confirmed that the EcoRI site has been successfully mutated by restriction digestion and sequencing (Data not shown).
a. Digestion of the targeting devices
As shown in Figures 3, 4 and 5 all the cuts made with the restriction enzymes seems to have the expected size, which helped us confirm for our constructs. We next sent our constructs to sequencing, which finally confirmed the correct assembly of them.
Figure 3. Restriction digestion of the construct GFP+ RuBisCO small.
Well 1: DNA ladder 1kb from Invitrogen. Well 2-6: C.GS digestion with SpeI and EcoRI. The expected sizes were 4562 and 2240.
Figure 4. Restriction digestion of the construct RuBisCO small+ GFP and pSB4K5. On well 1 DNA ladder 1kb from Invitrogen.
Well 2-3: pSB4K5 digested with DpnI. The expected sizes were 3149, 2558, 1623, 1440, 1363, 1170, 1145 & 570. Well 4-5: C.SG digested with PstI and EcoRI. The expected size were 4576 and 2240.
Figure 5. Restriction digestion of the construct GFP+ RuBisCO large and RuBisCO large+GFP.
On well 1 & 6 DNA ladder 1kb from Invitrogen. Well 2-4: C.GL digested with EcoRI. The expected sizes were 6127 and 3398. Well 5: C.LG digested with EcoRI. The expected sizes were 5383 and 3398.
b. GFP expression
To further study the functionality of our devices, we next test the expression of fluorescence. The devices included the different subunits of the RuBisCO attached with superfolder green fluorescent protein under the regulation of an inducible promoter sensitive to L-arabinose. We induced bacteria transformed with our construct and observed the presence of fluorescence (Figure 6). We modulated the expression of GFP by inducing with different concentrations of L-arabinose and found that with a 0.1mM the pattern of expression was focalized in the presence of Carboxysomes, which it’s consistent with the targeting functionality.
Figure 6. GFP expression.
E. coli was transformed with our constructs were induced with 0.1mM of L-arabinose. The GFP expression was observed under the epifluorescence microscope with a magnification of 100X, the brightfield image and the GFP filtered are shown.
3. Co-localization
The final goal of our constructs it is the delivery of the GFP into the Carboxysome, to observe this we co-transformed TOP10 with both the Carboxysome BioBrick (Bba_K1113100) + RFP (Bba_E1010) and our constructs to observe if they colocalized.
According to the results obtained with the Confocal microscopy, we observed co localization with both the C.LG and C.SG. However, after counting the number of bacteria while taking into consideration the following parameters:
Filamentous form of the bacteria
One or more red clusters
One or more green clusters
Complete merge of the green and red clusters
The construct that gave us the better merge was the large subunit of RuBisCO with the GFP in the carboxyl terminus.
Figure 7. Confocal microscopy.
We observed the colocalization of RFP and GFP of induced bacteria with both the Carboxysome BioBrick (Bba_K1113100) + RFP (Bba_E1010) and our strategies induced with IPTG and L-Arabinose.
Table 1.Quantification of co-localization.
The counting was made considering all the number of bacteria that shown a merge of the red and green fluorescence from figure 7.
Filamentous form of the bacteria
One or more red clusters
One or more green clusters
Complete merge of the green and red clusters
The construct that gave us the better merge was the large subunit of RuBisCO with the GFP in the carboxyl terminus.
