Team:Buenos Aires/Results/Bb1

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Revision as of 17:40, 26 September 2012

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  • Synthetic ecology
  • Overview
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  • Independent Population Control
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• Results and Devices
  • Strains characterization
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  • Fluorescence screening
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  • Auxotrophy confirmation
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  • Revertant control
  • Trp basal production
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  • BBs design
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  Data Page

Devices Design for Synthetic Ecology

In order for the cross-feeding scheme to work we need each strain to export the amino acid they produce (either Histidine or Tryptophan). To achieve this we created a devices design to secrete to the medium an His (or Trp) rich peptides.

Due to time and resources limitation, we decided to simplify the construction (assembly) process as much as possible. The devices were ordered as a whole (as gBlocks gene fragments) instead of obtaining each constitutive part and then assembling them. Although this goes against the standard part base approach, it saved both time and money. We plan to obtain each constitutive part from the devices by PCR with suffix/prefix containing primers, as a contribution to the registry.

We decided to use yeast expression plasmids with repressible or constitutive promoters, to drive the expression of our devices. This decision was taken because such plasmids were readily available to us, they had adequate selection markers and they are a fairly standard approach in yeast genetics.

The design of the devices with their constitutive parts is schematically represented in Figure 1.

Figure 1: Schematic representation of the (generic) devices for the cross-feeding design. RS: Restriction Sites, Prefix, Suffix: BioBrick standard, Signal: Secretion signal peptide. The ORF is highlighted in violet.

Each device has the following parts:

• Prefix of the BioBrick standard.
• Kozak consensus sequence for initiation of translation.
• Signal peptide that targets the product of the gene for secretion.
• Trojan peptide to increase internalization in target cell.
• Payload: this is the exported amino acid rich domain of the protein.
• Suffix of the BioBrick standard.

Note: Although the prefix and suffix are not considered part of the device, they were included in the ordered gene fragment for future assembly.

In addition, we included convenient restriction sites (RS) for directional cloning into the yeast expression vectors (RS1 and RS3). RS2 will allow easy removal of the sequence coding for the trojan peptide, by restriction and re-ligation.

Assembly Standard Prefix and Suffix

Because the entire ORF is contained within the prefix and suffix, no care for in-frame assembly has to be taken. We decided to use the original RFC10 BioBrick standard.

Figure2: RFC10 BioBrick standard

BioBricks

In order for the cross-feeding scheme to work we need each strain to export the amino acid they produce (either Histidine or Tryptophan). To achieve this we created a devices design to secrete to the medium an His (or Trp) rich peptides.

The basic structure of the devices with their constitutive parts is schematically represented in the next figure:

FIGURA SIMPLE

• Kozak consensus sequence for initiation of translation.
• Signal peptide that targets the product of the gene for secretion.
• Trojan peptide to increase internalization in target cell.
• Payload: this is the exported amino acid rich domain of the protein.

JUSTIFICACION DE PORQUE EL DISPOSTIVO NO TIENE UN PROMOTOR??? We decided to use yeast expression plasmids with repressible or constitutive promoters, to drive the expression of our devices. This decision was taken because such plasmids were readily available to us, they had adequate selection markers and they are a fairly standard approach in yeast genetics.

We built 2 His-export devices, and 2 Trp-export devices:

• His-export I (<partinfo>BBa_K792009</partinfo>)
• Trp export I (<partinfo>BBa_K792010</partinfo>)
• His-export II (<partinfo>BBa_K792011</partinfo>)
• Trp export II (<partinfo>BBa_K792012</partinfo>)

To achive this, we had to create several new basic biobricks:

• basicBB1
• basicBB2

Details about how we create these new basic biobricks can be found in the next sections. More details can be found in their registry entries also.

The next table summaries each export device composition.

Kozak Sequence

Signal Peptide

The signal peptides target proteins for secretion, effectively exporting the payload. This peptides are cleaved once the protein is in the lumen of the ER, so they won't have any further relevance.

Part <partinfo>BBa_K416003</partinfo> of the registry codes for a yeast signal peptide, based on [Clements 1991]. As an alternative we designed a second part (<partinfo>BBa_K792002</partinfo>) coding for another signal papetide, from the yeast α-mating factor gene ([http://www.yeastgenome.org/cgi-bin/locus.fpl?dbid=S000006108 MFα1]) [Waters et al 1987]. This part is likely to work well when combined with the Kozak sequence from the same gene (<partinfo>BBa_K792001</partinfo>, see above), as the natural 5' end of the MFα1 transcript is reconstituted. Also, because it is a yeast gene, it can be used as is, without any optimization.

The DNA sequence are shown in the following table.

Trojan peptide

Trojan peptides are short (15aa) sequences that penetrate through the plasma membrane inside the cell without the need of any receptor or endocitosis process [Derossi 1998]. We want to use them to increase the efficiency with which the payload enters the target cell. Ideally, they should not contain Trp or His, as those are the relevant amino acids for exportation. Two good candidates are the penetratin from the HIV TAT protein, and polyarginine [Jones et al 2005].

This proteins are not from yeast, so we needed to retro-translate them and codon-optimize them for expression in yeast. To do this we used the R package [http://www.bioconductor.org/packages/2.10/bioc/html/GeneGA.html geneGA], that takes into account codon usage and messenger secondary structure in the optimization process.

Payloads

The payloads are the elements of the synthetic gene that code for the “amino acid rich” region of the secreted protein. By “a.a. rich” we mean, rich in the amino acid we want to export, Trp or His in our case. These domains should be soluble enough not to cause precipitation of the protein, and should be relatively stable not to be degraded before they are actually secreted from the cell.

PolyHa is Clontech’s His tag sequence. TrpZipper2 is a small peptide that folds into a beta-hairpin secondary structure. The indole rings of the Trp form a hydrophobic core. The protein is water soluble and monomeric [Cochran 2001].

PolyHb and PolyWb were desing taking into acount the following consideration:

1. avoided repeating the same residue in tandem to minimize local tRNA depletion
2. avoided tryptophan in tandem because of their low solubility
3. we included glycine to avoid the formation of rigid structures
4. included acidic and basic amino acids to increase solubility

Retro-translating and optimizing for yeast (as explained above), we obtained the final sequences (see the registry for more details).

ORFs Arrangements

We are planning to synthesize 4 genes with different combinations of signal peptides, trojans and payloads. We want these genes to be immediately functional (no cloning necessary), but we also want o to have the possibility to recombine the parts in different arrangements. The 4 ORFs we will synthesize are described in Table 8.

Table 8: Arrangement of the synthesized ORFs

This arrangement would allow to compare ORF1 with ORF2, and ORF3 with ORF4.

Expression Plasmids

To determine which restriction sites to use for cloning (RS1 and RS3 in Figure 1), we need to know the MCS of the expression plasmids we are going to use.

One of these plasmids will probably of the pCM180-5 series, which are centromeric plasmids with TRP1 marker, and with a doxycycline repressible promoter [Gari et al 1996].

The Multiple Cloning Site of these vectors is shown in Figure 3.

Figure 3. Multiple Cloning Site (MCS) of the pCM180 series plasmids.

The other plasmid we might use is pEG202, with a 2 ori, HIS3 marker and a constitutive promoter (PADH1).

Figure 4: pEG202 MCS sequence and restriction sites.

Comparing Figure 3 and Figure 4, BamHI and NotI appear in both MCS in the same order, so they are good candidates for RS1 and RS3 (Figure 1) respectively.

We will probably need to clone the construct in a general purpose plasmid for manipulation. For instance, to remove the trojan we need to clone the construct into a plasmid, cut it with the RE of RS2, precipitate the DNA (to get rid of the trojan fragment), and religate. For this to work we have to make sure that there is no RS2 in the vector. A common vector for this is pBluescript, wich has the MCS shown in Figure 5.

Figure 5: pBluescript Multiple Cloning Site

Restriction Enzymes

The restriction sites used for RS1-3 have to be different from the ones used in the BioBricks standard. The standard RE for BioBricks are EcoRI, NotI, XbaI, SpeI and PstI.

Unfortunately, NotI was our candidate for RS3, so we have a problem here. There are different solutions. We can either use two restriction sites instead of RS3, one for each plasmid, or we can change the BioBrick standard to something like RFC[21] (Berkeley standard) that has no NotI restriction site.

An other option would be to use the restriction sites in the prefix and suffix to clone the construct into the expression plasmid. This is appealing because we don´t need new REs. Anyhow we would need to include a RS in the 5’ end to be able to directionally clone into pCM180. The new design would look something like Figure 6.

Figure 6: Alternative scheme for the restriction sites

If we make RS4 a BamHI site, we can directionally clone the construct into both plasmids (pCM180 and pEG202 ) by cutting with BamHI and NotI. In this design we would not need RS1 and RS3, but we can include them just in case we need to clone them into an other vector.

Regarding RS2 we need a restriction enzyme that produces cohesive ends, codes for acceptable amino acids, is easily available and not used in an other part of the construct. Some candidate RE are listed in Table 9.

Table 9. Candidate restriction enzymes for RS2

Probably any of them will work, but the trojan peptide needs to be basic so the HindIII site looks better suited. If we want to remove the trojan, we will have to clone the construct into a vector with no HindIII site. One way to do this is to clone it into pBluescript in such a way that the HindIII restriction site of the MCS is removed.

Looking at Figure 5 we can see that if we cut pBluescript with XhoI and PstI, the HindIII site is removed. If we make RS1 -> XhoI (which is easily available) we can cut the construct with these same enzymes and directionally clone it into pBluescript.

Most likely we wont use RS3, but we can assign it a restriction site just in case. For example NcoI could be used instead of NotI to do the directional cloning into pEG202.

Table 10. Assignation of restriction enzymes to the different restriction sites.

When we order the construct we will probably have to specify in which vector we want it shipped. RS3 (and perhaps RS1) could be changed depending on this vector. For example we could order the gene in pBluescript, in which case it might be convenient to make RS3 -> SacI. For other plasmids other RS might be needed, but if possible it would be convenient to retain RS1 -> XhoI that allows us to clone the gene into pBluescript as described above.