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Team:Amsterdam/Biobricks/Basic Parts
From 2011.igem.org
Contents |
Basic Parts
Three different types of basic parts were used to construct our CryoBricks: promoters, ribosome binding sites (RBSes) and coding regions. This page contains information on their purpose in the project. Emphasis is placed on the reason why we selected these particular parts, and in case of the coding regions, the biological function of the encoded protein.
Promoters
Several criteria were considered in selecting promoters for use in our CryoBricks. An ideal promoter would be: available from the parts registry, so that we can easily obtain and incorporate it; active at low temperatures, so that it works when we need it to; and positively regulated, so it won't be active unless we want it to. A search for promoters that matched one or more of these criteria resulted in the following selection:
pLacI
The Lac operon's promoter, which is often used to induce expression of proteins from plasmids artificially transformed into E. coli. Literature has numerous examples of this promoter being used to (over)express proteins at low temperatures, and it's available from the registry as [http://partsregistry.org/Part:BBa_R0010 BBa_R0010]. Unfortunately, it's not positively regulated, but its activity can be controlled to a certain extent. It's negatively regulated by the LacI protein, and as such can be stimulated by repressing LacI with IPTG or lactose, but it will be active constitutively in strains that don't express LacI.
pBAD
The promoter of the araBAD operon. Its biological function is similar to that of pLacI; in its natural context, it activates transcription of enzymes involved with the consumption of a carbon source, when this carbon source is available to the cell. Its activity is mediated by the AraC protein, which either activates of represses expression, depending on the concentration of L-arabinose. The pBAD promoter is available from the registry as [http://partsregistry.org/Part:BBa_I13453 BBa_I13453], but team British Columbia 2009 derived two new promoters from this part, responding either more or less strongly to the presence of arabinose (Stronger response: [http://partsregistry.org/Part:BBa_K206000 BBa_K206000], weaker response: [http://partsregistry.org/Part:BBa_K206001 BBa_K206001]). These pBAD derived promoters were selected because of their different arabinose sensitivity. This difference can facilitate finetuning the expression levels of proteins while using a single inducer, which can be further tweaked by combining the promoters with different RBSes.
hybB
The promoter of the hydrogenase II operon. It's also known as the coldshock promoter, because it's inactive at 37°C and activates at temperatures below 30°C. Using this promoter would allow us to only have our cells express CryoBricks when E. coli actually benefits from this, but makes inducing expression in a dose-dependent manner impossible, as it's not induced by a molecule we add. It enables CryoBrick expression to be directed on 'autopilot', which has pros (for example, in large-scale industrial applications) and cons (in case CryoBricks comprising such a promoter cross over to an uncontrolled environment, be it by accident or design). The registry submission of this brick, [http://partsregistry.org/Part:BBa_J45503 BBa_J45503], never made it past the planning stage, but the brick is incorporated in several composite parts.
Final selection
All selected promoters fulfilled the availability criterion, being submitted to the registry, but hybB was only available as part of an assembled construct. The construct was ordered and preparations were made to PCR it out of this construct, but constraints on time and safety considerations caused us to decide against using it in the end.
The project was supposed to include a characterisation experiment to quantify the remaining promoters' strengths at low temperatures. Unfortunately, technical difficulties with cloning, and subsequent assembly of the reporter constructs required for this, made such characterisation infeasible. Even though we couldn't confirm pLacI and pBAD's activity at low temperatures, it was decided to construct our CryoBricks with both promoters. Of the available pBAD promoters, we selected the one that responded most strongly to arabinose ([http://partsregistry.org/Part:BBa_K206000 BBa_K206000]), because with it, the maximum expression level is highest, but any of the expression levels attainable with the related promoters can be achieved as well, given a finely tuned concentration of arabinose.
RBSes
Similar to our promoter selection criteria, the ideal RBS for our CryoBricks should be active at low temperatures, and available from the registry. We suspect temperature may have quite a drastic impact on how an RBS functions; RBSes form structures in mRNA that mediate ribosome binding, but thermodynamics govern the dynamicity (and stability) of such structures. Literature study revealed only very little information about how temperature affects the activity of RBSes. To fill this gap in the knowledgebase, we planned to characterise some of the RBSes available from the registry, using reporter constructs to measure their strength at low temperatures. Unfortunately, technical difficulties and tight deadlines impaired these plans as they did with the plans to characterise promoter strengths.
Forced to omit characterising the strength of different RBSes at low temperatures, we decided to take our chances with the RBS team Warsaw 2010 defined as their standard against which to compare other RBS strengths ([http://partsregistry.org/Part:BBa_B0034 BBa_B0034]). Running on the crude assumption low temperatures affect all RBSes in an equivalent manner, we reasoned this RBS - which is relatively strong compared to other RBSes at 'normal' temperatures - should be relatively strong at low temperatures too.
